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The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand. The visible spectral properties revert to that of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous- nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450. [ABSTRACT FROM AUTHOR]

The reactions of fully reduced and mixed-valence bovine heart cytochrome c oxidase with dioxygen have been reinvestigated in the absence and presence of metal ions (Zn2+, Ni2+, and CD2+) by time-resolved optical absorption spectroscopy using the CO flow-flash technique. The time-resolved data were recorded on a microsecond to millisecond time scale at 442, 610, and 820 nm and subjected to quantitative amplitude analysis based on a conventional unidirectional sequential mechanism. The amplitudes of the sequential intermediates are derived from the absorbance changes associated with the different exponentials and from the kinetic equations of the sequential scheme. The general relationship between the pre-exponential factors and the absorbance of the successive intermediates in the sequential scheme is presented. A comparison of the experimental amplitudes of the individual intermediates with the model amplitudes at the three wavelengths indicates that the low spin heme a is incompletely oxidized during the formation of the sequential PR intermediate (PR,s). The conversion of the sequential F intermediate to the oxidized enzyme occurs on two millisecond time scales. The amplitude analysis of the single- wavelength data does not support the conventional sequential mechanism for the reduction of dioxygen to water catalyzed by cytochrome c oxidase. [ABSTRACT FROM AUTHOR]

Cytochrome c nitrite reductase is a dimeric decaheme-containing enzyme that catalyzes the reduction of nitrite to ammonium. The contrasting effects of two inhibitors on the activity of this enzyme have been revealed, and defined, by protein film voltammetry (PFV). Azide inhibition is rapid and reversible. Variation of the catalytic current magnitude describes mixed inhibition in which azide binds to the Michaelis complex (KdM ∼ 40 mM) with a lower affinity than to the enzyme alone (KdE ∼ 15 mM) and leads to complete inhibition of enzyme activity. The position of the catalytic wave reports tighter binding of azide when the active site is oxidized (KdO ∼39 μM) than when it is reduced. By contrast, binding and release of cyanide are sluggish. The higher affinity of cyanide for reduced versus oxidized forms of nitrite reductase is immediately revealed, as is the presence of two sites for cyanide binding and inhibition of the enzyme. Formation of the monocyano complex by reduction of the enzyme followed by a "rapid" scan to high potentials captures the activity-potential profile of this enzyme form and shows it to be distinct from that of the uninhibited enzyme. The biscyano complex is inactive. These studies demonstrate the complexity that can be associated with inhibitor binding to redox enzymes and illustrate how PFV readily captures and deconvolves this complexity through its impact on the catalytic properties of the enzyme. [ABSTRACT FROM AUTHOR]

Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro. The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria. The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria. Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da. SoxF catalyzed sulfide- dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a kcat of 3.9 s-1, a Km of 2.3 μM for sulfide, and a Km of 116 μM for cytochrome c, as determined by nonlinear regression analysis. The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product. Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (Ki = 1.3 μM) and inactivated by sulfite. Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92%. Redox titrations in the infrared spectral range from 1800 to 1200 cm-1 and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 ± 10 mY versus Ag/AgCl at pH 7.5 and -440 ± 20 mV versus Ag/AgCl at pH 6.0 (-232 mY versus SHE') and a transfer of 1.9 electrons. Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein. Furthermore, an activation/variation of SoxF during the redox cycles is observed. This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity. [ABSTRACT FROM AUTHOR]

Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a Kd of 16.4 μM at 25 °C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and ¹H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudo azurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 Å. This is consistent with the results of ¹H NMR spectroscopy, which showed that pseudo azurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme. [ABSTRACT FROM AUTHOR]

The kinetics of charge recombination following photoexcitation by a laser pulse have been analyzed in the reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides. In RC-LH1 core complexes isolated from photosynthetically grown cells P+QB- recombines with an average rate constant, (k) &app; 0.3 s-1, more than three times smaller than that measured in RC deprived of the LH1 ((k) &app; 1 s-1). A comparable, slowed recombination kinetics is observed in RC-LH1 complexes purified from a pufX-deleted strain. Slowing of the charge recombination kinetics is even more pronounced in RC-LH1 complexes isolated from wild-type semiaerobically grown cells ((k) &app; 0.2 s-1). Since the kinetics of P+QA- recombination is unaffected by the presence of the antenna, the P+QB- state appears to be energetically stabilized in core complexes. Determinations of the ubiquinone-10 (UQ10) complement associated with the purified RC-LH1 complexes always yield UQ10/RC ratios larger than 10. These quinone molecules are functionally coupled to the RC-LH1 complex, as judged from the extent of exogenous cytochrome C2 rapidly oxidized under continuous light excitation. Analysis of P+QB- recombination, based on a kinetic model which considers fast quinone equilibrium at the QB binding site, indicates that the slowing down of charge recombination kinetics observed in RC-LH1 complexes cannot be explained solely by a quinone concentration effect arid suggests that stabilization of the light-induced charge separation is predominantly due to interaction of the QB site with the LH1 complex. The high UQ10 complements detected in RC-LH1 core complexes, but not in purified light- harvesting complex 2 and in RC, are proposed to reflect an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer. [ABSTRACT FROM AUTHOR]

The H2O oxidizing domain of the cyanobacterial photosystem II (PSII) complex contains a low potential, c-type cytochrome termed C550 that is essential for the in vivo stability of the PSII complex. A mutant lacking cytochrome C550 (ΔpsbV) in Synechocystis sp. PCC6803 has been further analyzed together with a construct in which the distal axial heme iron ligand, histidine 92, has been substituted with a methionine (C550-H92M). Heme staining of SDS-PAGE showed that the C550-H92M mutation did not disturb the accumulation and heme-binding properties of the cytochrome. In ΔpsbV cells, the number of charge separating PSII centers was estimated to be 56% of the wild type, but of the existing centers, 33% lacked photooxidizable Mn ions. C550-H92M did not discernibly affect the intrinsic PSII electron-transfer kinetics compared to the wild type nor did it exhibit a significant fraction of centers lacking photooxidizable Mn; however, the number of charge separating PSII centers in mutant cells was 69% of the wild type. C550-H92M lost photoautotrophic growth ability in the absence of Ca2+, but its growth was not affected by depletion of Cl-, which differs from ΔpsbV. Taken together, the results suggest that in the absence of cytochrome C550 electron transfer on the donor side is retarded perhaps at the level of Yz to P680+ transfer, the heme ligand. His92 is not absolutely required for assembly of functional PSII centers; however, replacement by methionine prevents normal accumulation of PSII centers in the thylakoid membranes and alters the Ca2+ requirement of PSII. The results are discussed in terms of current understanding of the Ca2+ site of PSII. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property involved in the regulation of respiration. It has been shown that the recombination kinetics of photolyzed NO with reduced CcO from Paracoccus denitrificans on the picosecond time scale depend strongly on the NO/enzyme stoichiometry and inferred that more than one NO can be accommodated by the active site, already at mildly suprastoichiometric NO concentrations. We have largely extended these studies by monitoring rebinding dynamics from the picosecond to the microsecond time scale, by performing parallel steady-state low-temperature electron paramagnetic resonance (EPR) characterizations on samples prepared similarly as for the optical experiments and comparing them with molecular-modeling results. A comparative study was performed on CcO ba3 from Thermus thermophilus, where two NO molecules cannot be copresent in the active site in the steady state because of its NO reductase activity. The kinetic results allow discrimination between different models of NO-dependent recombination and show that the overall NO escape probability out of the protein is high when only one NO is bound to CcO aa3, whereas strong rebinding on the 15-ns time scale was observed for CcO ba3. The EPR characterizations show similar results for aa3 at substoichiometric NO/enzyme ratios and for ba3, indicating formation of a 6-coordinate heme--NO complex. The presence of a second NO molecule in the aa3 active site strongly modifies the heme--NO EPR spectrum and can be rationalized by a rotation of the Fe-N-O plane with respect to the histidine that coordinates the heme iron. This proposal is supported by molecular-modeling studies that indicate a ∼63° rotation of heme-bound NO upon binding of a second NO to the close-lying copper center CuB. It is argued that the second NO binds to CuB. [ABSTRACT FROM AUTHOR]

The thioredoxin fold is found in proteins that serve a wide variety of functions. Among these are peroxiredoxins, which catalyze the reduction of hydrogen peroxide and alkyl peroxides. Although the common structural fold shared by thioredoxins and peroxiredoxins suggests the possibility that they have evolved from a common progenitor, it has been difficult to examine this hypothesis in depth because pairwise sequence identities between proteins in these two superfamilies are statistically insignificant. Using the Shotgun program, we have found that sequences of reductases involved in maturation of cytochromes in certain bacteria bridge the sequences of thioredoxins and peroxiredoxins. Analysis of motifs found in a divergent set of thioredoxins, cytochrome maturation proteins, and peroxiredoxins provides further support for an evolutionary relationship between these proteins. Within the conserved motifs are specific residues that are characteristic of individual protein classes, and therefore are likely to be involved in the specific functions of those classes. We have used this information, in combination with existing structural and functional information, to gain new insight into the structure-function relationships in these proteins and to construct a model for the emergence of peroxiredoxins from a thioredoxin-like ancestor. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase requires multiple heme and copper cofactors to catalyze the reduction of molecular oxygen to water. Although significant progress has been made in understanding the transport and incorporation of the copper ions, considerably less is known about the trafficking and insertion of the heme cofactors. Heme O synthase (HOS) and heme A synthase (HAS) from Rhodobacter sphaeroides (Cox10 and Cox15, respectively) and Bacillus subtilis (CtaB and CtaA, respectively) have been cloned and expressed in Escherichia coli. Our results demonstrate that HOS copurifies with HAS and that HAS copurifies with HOS, indicating that HOS and HAS interact and may form a physiologically relevant complex in vivo. Consistent with this hypothesis, the presence of HAS alters the total level of farnesylated hemes, providing further evidence that HOS and HAS interact. Our current working model is that HOS and HAS form a complex and that heme O is transferred directly from HOS to HAS. Because of the strong sequence similarity and evolutionary relationship between R. sphaeroides and mitochondria, our data suggest that this complex may form in eukaryotes as well. [ABSTRACT FROM AUTHOR]

Bacillus azotofonnans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique men aquinol-dependent nitric oxide reductase (qCuANOR) with a CuA center in its small subunit. The qCuANOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the Em for all is close to 150 mV. Only one of these c cytochromes, which exhibits an a-band maximum at 551 nm, acts as a direct electron donor to qCuANOR. Further investigation demonstrated that this cytochrome c551 possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c551 is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCuANOR. The ability of qCUANOR to accept electrons from both menaquinol and cytochrome c551 might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus den itnflcans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c551, is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b6f complex serving the bioenergetic needs of the organism. [ABSTRACT FROM AUTHOR]

Several studies have found millisecond protein folding reactions to be controlled by the viscosity of the solvent: Reducing the viscosity allows folding to accelerate. In the limit of very low solvent viscosity, however, one expects a different behavior. Internal interactions, occurring within the solvent-excluded interior of a compact molecule, should impose a solvent-independent upper limit to folding speed once the bulk diffusional motions become sufficiently rapid. Why has this not been observed? We have studied the effect of solvent viscosity on the folding of cytochrome c from a highly compact, late-stage intermediate configuration. Although the folding rate accelerates as the viscosity declines, it tends toward a finite limiting value ∼105 s-1 as the viscosity tends toward zero. This limiting rate is independent of the cosolutes used to adjust solvent friction. Therefore, interactions within the interior of a compact denatured polypeptide can limit the folding rate, but the limiting time scale is very fast. It is only observable when the solvent- controlled stages of folding are exceedingly rapid or else absent. Interestingly, we find a very strong temperature dependence in these "internal friction"-controlled dynamics, indicating a large energy scale for the interactions that govern reconfiguration within compact, near-native states of a protein. [ABSTRACT FROM AUTHOR]

Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction [Green, A. J., Rivers, S. L., Cheesman, M., Reid, G. A., Quaroni, L. G., Macdonald, I. D. G., Chapman, S. K., and Munro, A. W. (2001) J. Biol. Inorg. Chem. 6, 523-533. Stok, J. E., and De Voss, J. J. (2000) Arch. Biochem. Biophys. 384, 351-360]. Here we show novel binding features of P450 BioI-specifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (Kd values in the range 0.1-160 μM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C(3) position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI = -330 ± 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 ± 2 mV; for palmitoleic acid-bound BioI = -199 ± 2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure. [ABSTRACT FROM AUTHOR]

The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressed in Escherichia coli and purified. DNA sequencing, mass spectrometry, and flavin-binding properties showed that both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively) and that an error in the published B. subtilis genome sequence had resulted in an altered reading frame and misassignment of YkuP as a long-chain flavodoxin. YkuN and YkuP were expressed in their blue (neutral semiquinone) forms and reoxidized to the quinone form during purification. Potentiometry confirmed the strong stabilization of the semiquinone form by both YkuN and YkuP (midpoint reduction potential for oxidized/semiquinone couple = -105 mV/-105 mV) with respect to the hydroquinone (midpoint reduction potential for semiquinone/hydroquinone couple = -382 mV/-377 mV). Apoflavodoxin forms were generated by trichloroacetic acid treatment. Circular dichroism studies indicated that flavin mononucleotide (FMN) binding led to considerable structural rearrangement for YkuP but not for YkuN. Both apoflavodoxins bound FMN but not riboflavin avidly, as expected for short-chain flavodoxins. Structural stability studies with the chaotrope guanidinium chloride revealed that there is moderate destabilization of secondary and tertiary structure on FMN removal from YkuN, but that YkuP apoflavodoxin has similar (or slightly higher) stability compared to the holoprotein. Differential scanning calorimetry reveals further differences in structural stability. YkuP has a lower melting temperature than YkuN, and its endotherm is composed of a single transition, while that for YkuN is biphasic. Optical and fluorimetric titrations with oxidized flavodoxins revealed strong affinity (Kd values consistently <5 μM) for their potential redox partner P450 BioI, YkuN showing tighter binding. Stopped-flow reduction studies indicated that the maximal electron-transfer rate (kred) to fatty acid-bound P450 BioI occurs from YkuN and YkuP at ∼ 2.5 s-1, considerably faster than from E. coli flavodoxin. Steady-state turnover with YkuN or YkuP, fatty acid-bound P450 BioI, and E. coli NADPH-flavodoxin reductase indicated that both flavodoxins supported lipid hydroxylation by P450 BioI with turnover rates of up to ∼ 100 min-1 with lauric acid as substrate. Interprotein electron transfer is a likely rate-limiting step. YkuN and YkuP supported monohydroxylation of lauric acid and myristic acid, but secondary oxygenation of the primary product was observed with both palmitic acid and palmitoleic acid as substrates. [ABSTRACT FROM AUTHOR]

Rieske proteins carry a redox-active iron-sulfur cluster, which is bound by two histidine and two cysteine side chains. The reduction potential of Rieske proteins depends on pH. This pH dependence can be described by two pKa values, which have been assigned to the two iron-coordinating histidines. Rieske proteins are commonly grouped into two major classes: Rieske proteins from quinol-oxidizing cytochrome bc complexes, in which the ligand histidines titrate in the physiological pH range, and bacterial ferredoxin Rieske proteins, in which the ligand histidines are protonated at physiological pH. In the study presented here, we have calculated pKa values of the cluster ligand histidines using a combined density functional theory/continuum electrostatics approach. Experimental pKa values for a bc-type and a ferredoxin Rieske protein could be reproduced. We could identify functionally important differences between the two proteins: hydrogen bonds toward the cluster, which are present in bc-type Rieske proteins, and negatively charged residues, which are present in ferredoxin Rieske proteins. We removed these differences by mutating the proteins in our calculations. The Rieske centers in the mutated proteins have very similar pKa values. We thus conclude that the studied structural differences are the main reason for the different pH-titration behavior of the proteins. Interestingly, the shift caused by neutralizing the negative charges in ferredoxin Rieske proteins is larger than the shift caused by removing the hydrogen bonds toward the cluster in bc-type Rieske proteins. [ABSTRACT FROM AUTHOR]

Expression of the truncated (lacking an N-terminal signal sequence) structural gene of Thermus thermophilus cytochrome C552 in the cytoplasm of Escherichia coli yields both dimeric (rC557) and monomeric (rC552) cytochrome c-like proteins [Keightley, J. A., et al. (1998) J. Biol. Chem. 273, 12006-12016], which form spontaneously without the involvement of cytochrome c maturation factors. Cytochrome rC557 is comprised of a dimer and has been structurally characterized [McRee, D., et al. (2001) J. Biol. Chem. 276,6537-6544]. Unexpectedly, the monomeric rC552 transforms spontaneously to a cytochrome- like chromophore having, in its reduced state, the Q00 transition (α-band) at 572 nm (therefore called p572). The X-ray crystallographic structure of rC552, at 1.41 Å resolution, shows that the 2-vinyl group of heme ring I is converted to a [heme-CO-CH2-S-CH2-Ca] conjugate with cysteine 11. Electron density maps obtained from isomorphous crystals of p572 at 1.61 Å resolution reveal that the 2-vinyl group has been oxidized to a formyl group. This explains the lower energy of the Q00 transition, the presence of a new, high-frequency band in the resonance Raman spectra at 1666 cm-1 for oxidized and at 1646 cm-1 for reduced samples, and the greatly altered, paramagnetically shifted ¹H NMR spectrum observed for this species. The overall process defines a novel mechanism for oxidation of the 2-vinyl group to a 2-formyl group and adds to the surprising array of chemical reactions that occur in the interaction of heme with the CXXCH sequence motif in apocytochromes c. [ABSTRACT FROM AUTHOR]

tert-Butyl acetylene (tBA) is a mechanism-based inactivator of cytochromes P450 2E1 and 2E1 T303A; however, the inactivation of the T303A mutant could be reversed by overnight dialysis. The inactivation of P450 2E1 T303A, but not the wild-type 2E1 enzyme, by tBA resulted in the formation of a novel reversible acetylene-iron spectral intermediate with an absorption maximum at 485 nm. The formation of this intermediate required oxygen and could be monitored spectrally with time. Although the alternate oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of wild-type P450 2131 by tBA in a reductase- and NADPH-free system, only tBHP supported the inactivation of the 2E1 T303A mutant. The losses in enzymatic activity occurred concomitantly with losses in the native P450 heme, which were accompanied by the formation of tBA-adducted heme products. The inactivations supported by tBHP and CHP were completely irreversible with overnight dialysis. Spectral binding constants (Ks) for the binding of tBA to the 2E1 P450s together with models of the enzymes with the acetylenic inactivator bound in the active site suggest that the T303A mutation results in increased hydrophobic interactions between tBA and nearby P450 residues, leading to a higher binding affinity for the acetylene compound in the mutant enzyme. Together, these data support a role for the highly conserved T303 residue in proton delivery to the active site of P450 2E1 and in the inactivation of the 2E1 P450s by small acetylenic compounds. [ABSTRACT FROM AUTHOR]

The roles of aromatic residues in redox regulation of cytochrome C3 were investigated by site-directed mutagenesis at every aromatic residue except for axial ligands (Phe20, Tyr43, Tyr65, Tyr66, His67, and Phe76). The mutations at Phe20 induced large chemical shift changes in the NMR signals for hemes 1 and 3, and large changes in the microscopic redox potentials of hemes 1 and 3. The NMR signals of the axial ligands of hemes 1 and 3 were also affected. Analysis of the nature of the mutations revealed that a hydrophobic environment and aromaticity are important for the reduction of the redox potentials of hemes 1 and 3, respectively. There was also a global effect. The replacement of Tyr66 with leucine induced chemical shift changes for heme 4, and changes in the microscopic redox potentials of heme 4. The mutations of Tyr65 induced changes in the chemical shifts and microscopic redox potentials for every heme, suggesting that Tyr65 stabilizes the global conformation, thereby reducing the redox potentials. In contrast, although the mutations of His67 and Phe76 caused chemical shift changes for heme 2, they did not affect its redox potentials, showing these residues are not important. All noncoordinated aromatic residues conserved in the cytochrome C3 subfamily with heme binding motifs CXXCH, CXXXXCH, CXXCH, and CXXXXCH (Phe20, Tyr43, and Tyr66) are involved in the π-π interaction, which causes a decrease in the redox potential of the interacting heme. The global effect can be attributed to either direct or indirect interactions among the four hemes in the cyclic architecture. [ABSTRACT FROM AUTHOR]

The spectral and redox properties are described for the wild-type and Y384F mutant forms of the flavoprotein component (PchF) of flavocytochrome, p-cresol methylhydroxylase (PCMH), and cytochrome-free PchF that harbor FAD analogues. The analogues are iso-FAD (8-demethyl-6-methyl- FAD), 6-amino-FAD (6-NH2-FAD), 6-bromo-FAD (6-Br-FAD), 8-nor- 8-chloro-FAD (8-Cl-FAD), and 5-deaza-5-carba-FAD (5-deaza-FAD), All of the analogues bound noncovalently and stoichiomethcally to cytochrome-free apo-PchF, and the resulting holoproteins had high affinity for the cytochrome subunit, PchC. Noncovalently bound FAD, 6-Br-FAD, or 6-NH2-FAD can be induced to bind covalently by exposing holo-PchF to PchC. The rate of this process and the redox potential of the noncovalently bound flavin may be correlated. In addition, the redox potential of each FAD analogue was higher when it was covalently bound than when noncovalently bound to PchF. Furthermore, the potential of a covalently bound or noncovalently bound FAD analogue increased on association of the corresponding holo-PchF with PchC, and the activity increased as the flavin's redox potential increased. It was discovered also that 4-hydroxybenzaldehyde, the final p-cresol oxidation product, is an efficient competitive inhibitor for substrate oxidation by PchF since it binds tightly to this protein when the flavin is oxidized, although it binds more loosely to the enzyme with reduced flavin. Finally, the energies of the charge-transfer bands for the interaction of bound flavin analogues with 4-Br-phenol (a substrate mimic) increased as the potential decreases, although a simple global correlation was not seen. This is the case because the energy is also a function of the redox properties of the bound mimic. The implications of these findings to covalent flavinylation and catalysis are discussed. [ABSTRACT FROM AUTHOR]

The complete genome sequence of the &odelta;-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes. Cytochrome c7, isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with Mr 9.6 kDa. This protein is involved in metal ion- reducing pathways and shares 56% sequence identity with a triheme cytochrome isolated from the closely related δ-proteobacterium Desulfuromonas acetoxidans (Dac7). In this work, two-dimensional NMR was used to monitor the heme core and the general folding in solution of the G. sulfurreducens triheme cytochrome c7 (PpcA). NMR signals obtained for the three hemes of PpcA at different stages of oxidation were cross-assigned to the crystal structure [Pokkuluri, P. R., Londer, Y. Y., Duke, N. E. C., Long, W. C., and Schiffer, M. (2004) Biochemistry 43, 849-859] using the complete network of chemical exchange connectivities, and the order in which each heme becomes oxidized was determined at pH 6.0 and 8.2. Redox titrations followed by visible spectroscopy were also performed in order to monitor the macroscopic redox behavior of PpcA. The results obtained showed that PpcA and Dac7 have different redox properties: (i) the order in which each heme becomes oxidized is different; (ii) the reduction potentials of the heme groups and the global redox behavior of PpcA are pH dependent (redox-Bohr effect) in the physiological pH range, which is not observed with Dac7. The differences observed in the redox behavior of PpcA and Dac7 may account for the different functions of these proteins and constitute an excellent example of how homologous proteins can perform different physiological functions. The redox titrations followed by visible spectroscopy of PpcA and two mutants of the conserved residue F15 (PpcAF15Y and PpcAF15W) lead to the conclusion that F15 modulates the redox behavior of PpcA, thus having an important physiological role. [ABSTRACT FROM AUTHOR]

Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfflydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme. [ABSTRACT FROM AUTHOR]

Redox transitions in the Rhodobacter capsulatus cytochrome bc1 complex were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with synchronous visible spectroscopy, in both the wild type and a cytochrome c1 point mutant, M 183K, in which the midpoint potential of heme was lowered from the wild-type value of 320 mV to 60 mV. Overall redox difference spectra of the wild type and M183K mutant were essentially identical, indicating that the mutation did not cause any major structural perturbation. Spectra were compared with data on the bovine bc1 complex, and tentative assignments of several bands could be made by comparison with available data on model compounds and crystallographic structures. The bacterial spectra showed contributions from ubiquinone that were much larger than in the bovine enzyme, arising from additional bound and adventitious ubiquinone. The M183K mutant enabled selective reduction of the iron-sulfur protein which in turn allowed the IR redox difference spectra of ISP and cytochrome c1 to be deconvoluted at high signal/noise ratios, and features of these spectra are interpreted in light of structural and mechanistic information. [ABSTRACT FROM AUTHOR]

Previously a K+-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K+ influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Tip191 cation radical is dependent on the occupancy of the engineered K+ site and that the Tip191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Tip191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 Å from the Tip radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Tip radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Tip radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Tip191 radical. [ABSTRACT FROM AUTHOR]

In this study a combined electrochemical and FTIR spectroscopic approach was applied to monitor the binding of stigmatellin, a Qo site inhibitor of the cytochrome bc1 complex from Saccharomyces cerevisiae. Natural stigmatellin A induced clear shifts in the redox-induced FTIR difference spectra. For data interpretation a stigmatellin derivative (UST) with the conjugated trienes replaced by an aliphatic tail was synthesized, and the carbonyl group shown in crystal structures to interact with His181, the [2Fe-2S] ligand of the Rieske, was specifically 13C labeled. Electrochemically induced ETIR difference spectra of the inhibitors in CH3OD were obtained and revealed signals characteristic for the oxidized and reduced forms of the labeled and unlabeled compounds. On the basis of signals from the inhibitors alone, the binding of the inhibitor to the bc1 complex was monitored. Direct evidence for the interaction of the carbonyl group with the protein was provided by the observed shift of the v(C=O) vibrational mode of about 10 cm-1. In addition, redox-dependent reorganizations of the protein were identified, including protonation changes of acidic residues at 1746 and 1734 cm-1. The conformational changes observed upon inhibitor binding are discussed with respect to the crystal structures and proposed mechanistic models [Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000) Structure 8, 669-684; Palsdottir, H., Lojero, C. G., Trumpower, B. L., and Hunte, C. (2003) J. Biol. Chem. 278, 31303-31311]. [ABSTRACT FROM AUTHOR]

The PrrBA two-component system in Rhodobacter sphaeroides 2.4.1, which is composed of the PrrB histidine kinase and the PrrA response regulator, controls the expression of all of the photosynthesis genes, either directly or indirectly, in response to changes in oxygen tension. In vivo under aerobic conditions it is the cbb3 cytochrome c oxidase which generates an inhibitory signal preventing the accumulation of activated PrrA. Using purified cbb3 cytochrome c oxidase, PnB, and PrrA, we demonstrate in vitro that the cbb3 oxidase inhibits PrrB activity by apparently increasing the intrinsic PrrB phosphatase activity, which dephosphorylates phosphorylated PrrA without alteration of the PrrB kinase activity. The transmembrane domain of PrrB is required for the enhancement of PrrB phosphatase activity by the cbb3 oxidase. Full-length PrrB has a significantly greater ability to phosphorylate PrrA than does truncated PrrB lacking the transmembrane domain. This is at least in part due to the lower autophosphorylation rate of the truncated PrrB relative to the full-length PrrB. This finding provides evidence that the sensing domain (transmembrane domain) of PrrB plays an important role not only in optimally sensing the state of the cbb3 oxidase but also in maintaining the correct conformation of PrrB, providing optimal autokinase activity. [ABSTRACT FROM AUTHOR]

Selected active site residues in substrate recognition sites (SRS) 1 and 5 of cytochrome P450 2C8 (CYP2C8) were mutated to the corresponding amino acids present in CYP2C9 to investigate the contribution of these positions to the unique substrate selectivity and regioselectivity of CYP2C8. The effects of mutations, singly and in combination, were assessed from changes in the kinetics of paclitaxel 6α-hydroxylation, a CYP2CS-specific pathway, and the tolylmethyl and ring hydroxylations of torsemide, a mixed CYP2C9/CYP2C8 substrate. Within SRS 1, the single mutation S1 14F abolished paclitaxel 6α-hydroxylation, while the I1 13V substitution resulted in modest parallel reductions in Km and Vmax. Mutations in SRS5 (viz., V362L, G365S, and V366L) reduced paclitaxel intrinsic clearance (Vmax/Km) by 88-100%. Torsemide is preferentially metabolized by CYP2C9, and it was anticipated that the mutations in CYP2C8 might increase activity. However, methyl and ring hydroxylation intrinsic clearances were either unchanged or decreased by the mutations, although hydroxylation regioselectivity was often altered relative to wild-type CYP2C8. The mutations significantly increased (28-968%) Km values for both torsemide methyl and ring hydroxylation but had variable effects on Vmax. The effects of the combined mutations in SRS 1, SRS5, and SRS 1 plus SRS5 were generally consistent with the changes produced by the separate mutations. Mutation of CYP2C8 at position 359 (S3591), a site of genetic polymorphism in CYP2C9, resulted in relatively minor changes in paclitaxel- and torsemide-hydroxylase activities. The results are consistent with multiple substrate binding orientations within the CYP2C8 active site and a differential contribution of active site residues to paclitaxel and torsemide binding and turnover. [ABSTRACT FROM AUTHOR]

All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case of Rhodobacter capsulatus cytochrome c2, the sixth heme ligand Met96 and up to 14 flanking residues (positions 88-100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region of Rhodobacter capsulatus cytochrome C2 and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93-100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole. [ABSTRACT FROM AUTHOR]

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-134121, we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q0 site of the cytochrome (cyt) b6f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b6f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q0 site. With a single DBMIB at the Q0 site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b6f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc1 complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt bL heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q0 pocket. With ≥ 2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISPB position of the chicken mitochondrial cyt bc1 complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b6f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc1 complex suggest that the soluble ISP domain of the spinach cyt b6f complex can rotate by at least 53°, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc6 complex and the M. laminosus and C. reinhardtii cyt b6f complexes. [ABSTRACT FROM AUTHOR]

The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity in cytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasing ionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, which fraction increases from 26% at 0.06 M to 75% at 1.2 M. This effect was associated with a considerable decrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinct positive cooperativity (S50 = 8.3 µM, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), n decreases to 1.2 (S50 = 3.2 µM) at I = 0.66 M. Increasing ionic strength also increases the distance between the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiency of FRET from 1-PB to the heme. The modification of Cys154 with 7-(diethylamino)-3-(4'-maleimidyl-phenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominent decrease in the cooperativity. The same effect was observed when Cys154 was substituted with isoleucine. Importantly, Cys154 is located at the C-terminal end of helix E and is surrounded by salt bridges formed by arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest that the binding of the first substrate molecule causes an important conformational transition in the P4SOeryF that facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociation or rearrangement of several salt bridges in the proximity of Cys154 and modulates accessibility and hydration of the heme pocket. [ABSTRACT FROM AUTHOR]

Our previous study revealed that human CYP24AI catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D3 and 1α,25(OH)2D3 as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y. and Inouye, K. (2000) Eur. J. Biochem. 267, 6158-61651. In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D3 and 25,26,27-trinor-23-ene-lα(OH)D3 as novel metabolites of 25(OH)D3 and 1α,25(OH)2D3, respectively. These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A 1 produces much more of these metabolites than does rat CYP24A1. We propose that the C24-C25 bond cleavage occurs by a unique reaction mechanism including radical rearrangement. Namely, after hydrogen abstraction of the C-23 position of 1α,25(OH)2D3, part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1α- (OH)D3, while a major part of them is converted into 1α,23,25(OH)3D3. Because the C24-C25 bond cleavage abolishes the binding affinity of I α,25(OH)D3 for the vitamin D receptor, this reaction is quite effective for inactivation of 1α,25(OH)D3. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of the cytochrome bσ complex disclosed the unexpected presence of a new heme ci [Stroebel, D., Choquet, Y., Popot, J.-L., and Picot, D. (2003) Nature 426, 413-418; Kurisu, G., Zhang, H., Smith, J. L., and Cramer, W. A. (2003) Science 302, 1009-1014]. Here we present a biochemical, spectroscopic, and mutagenesis study of this unusual heme binding in Chlamydomonas reinhardtii. As predicted by the structure data, we identify a Cys35-containing proteolytic fragment (Tyr25-Lys111) from cytochrome b6 as a peptide that covalently binds a heme. Resonance Raman spectra of cyt bσf complexes show particular frequencies in V2, V3, 1)4, and V8 regions that identify this extra heme as a ferrous c'-like heme under a five-coordinated high-spin state. The set of frequencies is consistent with a coordination by either a water molecule or a hydroxide ion. Other changes in resonance Raman bands, observed in the mid- and low-frequency regions, point to a modification in conformation and/or environment of at least one b heme methyl and/or propionate group. Site-directed mutagenesis of apocytochrome b6, leading to a Cys35Val substitution, generates Chlamydomonas strains that are unable to assemble cytochrome bσf complexes. On the basis of the mutant phenotype, we discuss the participation, in the covalent binding of heme ci, of the nuclear CCB factors that we identified previously as controlling the apo to holo conversion of cytochrome b6 [Kuras, R., de Vitry, C., Choquet, Y., Girard-Bascou, J., Culler, D., Büschlen, S., Merchant, S., and Wollman, F.-A. (1997) J. Biol. Chem. 272, 32427-32435]. [ABSTRACT FROM AUTHOR]

The diflavin reductases exemplified by mammalian cytochrome P450 reductase catalyze NADPH dehydrogenation and electron transfer to an associated monooxygenase. It has recently been proposed that double occupancy of the NADPH dehydrogenation site inhibits the NADPH to FAD hydride transfer step in this series of enzymes. This has important implications for the mechanism of enzyme turnover. However, the conclusions are drawn from a series of pre-steady-state stopped-flow experiments in which the data analysis and interpretation are flawed. Recent data published for P450-BM3 reductase show a decrease in the rate constant for pre-steady-state flavin oxidation with increasing NADP+ concentration. This is interpreted as evidence of inhibition by multiple substrate binding. A detailed reanalysis shows that the data are in fact consistent with a simple single-binding-site model in which reversible hydride transfer causes the observed effect. Data for the related systems are also discussed. [ABSTRACT FROM AUTHOR]

The mechanism of the dioxygen (O2) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy. The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O2-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms. The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms. The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after 1 ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o. In contrast, the enzyme without the bound ubiquinone-8 (ΔUbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the F1 intermediate possessing ferryl--oxo heme o, cupric CuB, and ferric heme b is converted to the FII intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the FII intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center. Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and CuB and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions. [ABSTRACT FROM AUTHOR]

Sequence alignment of the Rieske iron-sulfur protein (ISP) of cytochrome bc1 complex from various sources reveals that bacterial ISPs contain an extra fragment. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with deletion or single- or multiple-alanine substitution at various positions of this fragment (residues 96-107) were generated and characterized. The ISPA(96- 107), ISP(96- 107)A, and ISP( 104- 107)A mutant cells, in which residues 96-107 of ISP are deleted, and residues 96-107 and 104-107 are substituted with alanine, respectively, do not grow photosynthetically and show no bc1 complex activity in intracytoplasmic membranes prepared from these mutant cells. The ISP(96-99)A, in which residues 96-99 are substituted with alanine, grows photosynthetically at a rate comparable to that of the complement cells, whereas ISP(100-103)A, in which residues 100-103 are substituted with alanine, has a longer lag period prior to photosynthetic growth. Chromatophores prepared from these two mutant cells have 48% and 9% of the bc1 activity found in the complement chromatophores. The loss (or decrease) of bc1 activity in these mutant membranes results from a lack (or decrease) of ISP in the membrane due to ISP protein instability and not from mutations affecting the assembly of cytochromes b and c1 into the membrane, the binding affinity of cytochrome b to cytochrome c1, or the ability of these two cytochromes to interact with ISP or subunit IV. The order of essentiality of residues in this fragment is residues 104-107 > residues 100-103 > residues 96-99. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) 1A2 is the major enzyme involved in the metabolism of 2-amino-3,5-dimethylimidazo[4,5-flquinoline (MeIQ) and other heterocyclic arylamines and their bioactivation to mutagens. Random mutant libraries of human P450 1A2, in which mutations were made throughout the entire open reading frame, were screened with Escherichia coli DJ3109pNM12, a strain designed to bioactivate MeIQ and detect mutagenicity of the products. Mutant clones with enhanced activity were confirmed using quantitative measurement of MeIQ N-hydroxylation. Three consecutive rounds of random mutagenesis and screening were performed and yielded a highly improved P450 1A2 mutant, SF513 (E225N/Q258H/G437D), with > 10-fold increased MeIQ activation based on the E. coli genotoxicity assay and 12-fold enhanced catalytic efficiency (k[Subcat]/K[Subm]) in steady-state N-hydroxylation assays done with isolated membrane fractions. SF513 displayed selectively enhanced activity for MeIQ compared to other heterocyclic arylamines. The enhanced catalytic activity was not attributed to changes in any of several individual steps examined, including substrate binding, total NADPH oxidation, or H[Sub2]O[Sub2] formation. Homology modeling based on an X-ray structure of rabbit P450 2C5 suggested that the E225N and Q258H mutations are located in the F-helix and G-helix, respectively, and that the G437D mutation is in the "meander" region, apparently rather distant from the substrate. In summary, the approach generated a mutant enzyme with selectively elevated activity for a single substrate, even to the extent of a difference of a single methyl group, and several mutations had interacting roles in the development of the selected mutant protein. [ABSTRACT FROM AUTHOR]

The structure of a cytochrome c[sub7] (PpcA) from Geobacter sulfurreducens was determined by X-ray diffraction at 1.45 Å resolution; the R factor is 18.2%. The protein contains a three-heme core that is surrounded by 71 amino acid residues. An unusual feature of this cytochrome is that it has 17 lysine residues, but only nine hydrophobic residues that are larger than alanine. The details of the structure are described and compared with those of cytochrome c[sub7] from Desulfuromonas acetoxidans and with cytochromes c[sub3]. The two cytochrome c[sub7] molecules have sequences that are 46% identical, but the arrangements of the hemes in the two structures differ; the rms deviation of all α-carbons is 2.5 Å. These cytochromes can reduce various metal ions. The reduction site of the chromate ion in D. acetoxidans is occupied by a sulfate ion in the crystal structure of PpcA. We identified four additional homologues of cytochrome c[sub7] in the G. sulfurreducens genome and three polymers of c[sub7]-type domains. Of the polymers, two have four repeats and one has nine repeats. On the basis of sequence alignments, one of the hemes in each of the cytochrome c[sub7]-type domains does not have the bis-histidine coordination. The packing of the molecules in the crystal structure of PpcA suggests that the polymers have an elongated conformation and might form a "nanowire". [ABSTRACT FROM AUTHOR]

The kinetics of electron transfer from reduced high-potential iron-sulfur protein (HiPIP) to the photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center (RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redox conditions by flash absorption spectroscopy. At ambient redox potential E[subh] = +200 mV, where only the high-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HP heme(s) follows second-order kinetics with rate constant k = (4.2 ± 0.2) 10[sup5] M[sup-1] s[sup-1] at low ionic strength. Upon increasing the ionic strength, k increases by a maximum factor of ca. 2 at 640 mM KCI. The role of Phe48, which lies on the external surface of HiPIP close to the [Fe[sub4]S[sub4]] cluster and presumably on the electron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis. Substitution of Phe48 with arginine, aspartate, and histidine completely prevents electron donation. Conversely, electron transfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate is decreased by more than 1 order of magnitude. These results suggest that Phe48 is located on a key protein surface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobic residue on the putative electron-transfer pathway plays a critical role. This conclusion was supported by protein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involves a docking site close to the LP heme farthest from the bacteriochlorophyll special pair. [ABSTRACT FROM AUTHOR]

The orientation of the g-tensors of the Rieske iron-sulfur protein subunit was determined in a single crystal of the bovine mitochondrial cytochrome bc[sub1] complex with stigmatellin in the Q[subo] quinol binding site. The g-tensor principal axes are skewed with respect to the Fe-Fe and S-S atom direction in the 2Fe2S cluster, which is allowed by the lack of rigorous symmetry of the cluster. The asymmetric unit in the crystal is the active dimer, and the g-tensor axes have slightly different orientations relative to the iron-sulfur cluster in the two halves of the dimer. The g ∼ 1.79 axis makes an average angle of 30° with respect to the Fe-Fe direction and the g ∼ 2.024 axis an average angle of 26° with respect to the S-S direction. This assignment of the g-tensor axis directions indicates that conformations of the Rieske protein are likely the same in the cytochrome bc[sub1] and b[sub6]f complexes and that the extent of motion of the Rieske head domain during the catalytic cycle has been highly conserved during evolution of these distantly related complexes. [ABSTRACT FROM AUTHOR]

Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6β-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (FE) content in ternary systems of PCIPEIPA or PC/PB! PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PB might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b[sub5] (b[sub5]) and apolipoprotein b[sub5] further enhanced the testosterone 6β-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PB induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PB are critical for the membrane binding and catalytic activity of CYP3A4. [ABSTRACT FROM AUTHOR]

CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrate including endogenous steroids and some drugs that coexist at the active site. To clarify the mechanism of the effect of endogenous steroids on the drug metabolism, the interaction between substrates, nevirapine (NVP) and carbamazepine (CBZ), and endogenous steroids was investigated by theoretical calculations. When the activities of NVP 2-hydroxylation and CBZ 10,11-epoxidation by expressed CYP3A4 were measured in the presence of steroids, NVP 2-hydroxylation was found to be remarkably increased by aldosterone and inhibited by estradiol. CBZ 10,11-epoxidation was increased by andros- tenedione. Three-dimensional computer modeling has shown that the active site of CYP3A4 is especially large, permitting access of two substrate molecules. The interactions between NVP and aldosterone and between CBZ and androstenedione were estimated by theoretical calculations assuming the substrate and steroids to be present in the active site at the same time. It was shown that NVP or CBZ would be stably fixed close to the oxygen atom at the sixth ligand of heme by interaction with steroids, suggesting that NVP and CBZ may be hydroxylated more easily due to the interaction with steroids. Estradiol was also expected to interact with NVP via a &pie;/&pie; interaction between a benzene ring, in which the NVP hydroxylation site is located, and a benzene ring of estradiol, suggested to inhibit the reaction. From these results, interactions between the substrate and endogenous steroids in the active site may change the activity of CYP3A4. [ABSTRACT FROM AUTHOR]

The interaction between bovine cytochrome b5 (cyt b5) and horse heart cytochrome c (cyt c) is investigated by NMR spectroscopy. Chemical shifts of cyt b5 backbone resonances and side chain methyl resonances were monitored as a function of cyt c concentration. The shifts are small but saturatable and indicate that the binding of cyt b5 with cyt c is in fast exchange. An equilibrium association constant of (6 ±3) × 10[SUP4] M[SUP-1] was obtained with lowera limit of 180 s[SUP-] for the dissociation rate of the complex. To resolve considerable ambiguities in the interpretation of the chemical shift mapping, [SUP15]SN relaxation experiments and cross-saturation experiments were used as alternative methods to map the cyt b5-cyt c binding interface. Results from the three experiments combined demonstrate that the conserved negatively charged region of cyt b5 surrounding the solvent-exposed heme edge is involved in the interaction with cyt c. [ABSTRACT FROM AUTHOR]

Cytochrome P450s in endoplasmic reticulum membranes function in the hydroxylation of exogenous and endogenous hydrophobic substrates concentrated in the membranes. The reactions require electron supplies from NADPH--cytochrome P450 reductase in the same membranes. The membranes play important roles in the reaction of cytochrome P450. The membrane topology of guinea pig P450 17α was investigated on the basis of the differences in reactivity to hydrophilic chemical modification reagents between those in the detergent-solubilized state and proteoliposomes. Recombinant guinea pig cytochrome P450 17α was purified from Escherichia coli and incorporated into liposome membranes. Lysine residues in the detergent-solubilized P450 l7α and in the proteoliposomes were acetylated with acetic anhydride at pH 9.0, and the acidic amino acid residues were conjugated with glycinamide at pH 5.0 by the aid of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The modifications were performed under conditions where the denatured form, P420, was not induced. The modified P450 17α's were digested by trypsin, and the molecular weights of the peptide fragments were determined by MALDI-TOF mass spectrometry. From the increase in the molecular weights of the peptides, the positions of modifications could be deduced. In the detergent-solubilized state, 11 lysine residues and 7 acidic amino acid residues were modified, among which lysine residues at positions 29, 59, 490, and 492 and acidic residues at 211, 212, and/or 216 were not modified in the proteoliposomes. Both the N- and C-terminal domains and the putative F--G loop were concluded to be in or near the membrane-binding domains of P450 17α. [ABSTRACT FROM AUTHOR]

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm[SUP-1]. The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm[SUP-1] band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O[SUB2] and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting. [ABSTRACT FROM AUTHOR]

The structure of the complex between cytochrome c[SUB2] (cyt) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides shows contacts between hydrophobic residues Tyr L162, Leu M191, and Val M192 on the RC and the surface of the cyt [Axelrod et al. (2002) J. Mol. Biol. 319, 501-515]. The role of these hydrophobic residues in binding and electron transfer was investigated by replacing them with Ala and other residues. Mutations of the hydrophobic residues generally resulted in relatively small changes in the second-order electron-transfer rate k[SUB2] (Brönsted coefficient, α = 0.15 ± 0.05) indicating that the transition state for association occurs before short-range hydrophobic contacts are established. Larger changes in k[SUB2], found in some cases, were attributed to a change in the second-order mechanism from a diffusion controlled regime to a rapidly reversible binding regime. The association constant, K[SUBA], of the cyt and the rate of electron transfer from the bound cyt, k[SUBe], were both decreased by mutation. Replacement of Tyr L162, Leu M191, or Val M192 by Ala decreased K[SUBA] and k[SUBe] by factors of 130, 10, 0.6, and 120, 9, 0.6, respectively. The largest changes were obtained by mutation of Tyr L162, showing that this residue plays a key role in both binding and electron transfer. The binding affinity, K[SUBA], and electron-transfer rate, k[SUBe], were strongly correlated, showing that changes of hydrophobic residues affect both binding and electron transfer. This correlation suggests that changes in distance across hydrophobic interprotein contacts have similar effects on both electron tunneling and binding interactions. [ABSTRACT FROM AUTHOR]

The known activity of cytochrome P450 46A1 (P450 46A1) is 24(S)-hydroxylation of cholesterol. This reaction produces biologically active oxysterol, 24(S)-hydroxycholesterol, and is also the first step in enzymatic degradation of cholesterol in the brain. We report here that P450 46A1 can further metabolize 24(S)-hydroxycholesterol, giving 24,25- and 24,27-dihydroxycholesterols in both the cell cultures transfected with P450 46A1 cDNA and the in vitro reconstituted system with recombinant enzyme. In addition, P450 46A1 was able to carry out side chain hydroxylations of two endogenous C27-steroids with and without a double bond between C5-C6 (7α-hydroxycholesterol and cholestanol, respectively) and introduce a hydroxyl group on the steroid nucleus of the C2 1-steroid hormones with the C4-C5 double bond (progesterone and testosterone). Also, P450 46A1 was found to metabolize xenobiotics carrying out dextromethorphan O- and N-demethylations, diclofenac 4'-hydroxylation, and phenacetin O-deethylation. Thus, substrate specificities of P450 46A1 are not limited to cholesterol and include a number of structurally diverse compounds. Activities of P450 46A1 suggest that, in addition to the involvement in cholesterol homeostasis in the brain, this enzyme may participate in metabolism of neurosteroids and drugs that can cross the blood-brain bather and are targeted to the central nervous system. [ABSTRACT FROM AUTHOR]

Methemoglobinemia, the first hereditary disease to be identified that involved an enzyme deficiency, has been ascribed to mutations in the enzyme cytochrome b[SUB5] reductase. A variety of defects in either the erythrocytic or microsomal forms of the enzyme have been identified that give rise to the type I or type II variant of the disease, respectively. The positions of the methemoglobinemia-causing mutations are scattered throughout the protein sequence, but the majority of the non truncated mutants that produce type II symptoms occur close to the flavin adenine dinucleotide (FAD) cofactor binding site. While X-ray structures have been determined for the soluble, flavin-containing diaphorase domains of the rat and pig enzymes, no X-ray or NMR structure has been described for the human enzyme or any of the methemoglobinemia variants. S 127P, a mutant that causes type II methemoglobinemia, was the first to be positively identified and have its spectroscopic and kinetic properties characterized that revealed altered nicotinamide adenine dinucleotide hydride (NADH) substrate binding behavior. To understand these changes at a structural level, we have determined the structure of the S127P mutant of rat cytochrome b5 reductase to 1.8 Å resolution, providing the first structural snapshot of a cytochrome b[SUB5] reductase mutant that causes methemoglobinemia. The high-resolution structure revealed that the adenosine diphosphate (ADP) moiety of the FAD prosthetic group is displaced into the corresponding ADP binding site of the physiological substrate, NADH, thus acting as a substrate inhibitor which is consistent with both the spectroscopic and kinetic data. [ABSTRACT FROM AUTHOR]

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and + 150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK[SUBa] values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pKa, the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q[SUB0] site of the cytochrome bc[SUB1] and b[SUB6]f complexes are discussed. [ABSTRACT FROM AUTHOR]

The cytochrome bc[SUB1] complex from Paracoccus denitrificans and soluble fragments of its cytochrome c[SUB1] and Rieske ISP subunits are characterized by a combined approach of protein electrochemistry and FTIR difference spectroscopy. The FTIR difference spectra provide information about alterations in the protein upon redox reactions: signals from the polypeptide backbone, from the cofactors, and from amino acid side chains. We describe typical modes for conformational changes in the polypeptide and contributions of different secondary structure elements. Signals attributed to the different cofactors can be presented on the basis of selected potential steps. Modes associated with bound quinone are identified by comparison with spectra of quinone in solution at 1656, 1642, and 1610 cm[SUP-1] and between 1494 and 1388 cm[SUP-1], as well as at 1288 and 1262 cm[SUP-1]. Signals originating from the quinone bound at the Q[SUB0] site can be distinguished. On the basis of the infrared data, the total quinone concentration is determined to be 2.6-3.3 quinones per monomer, depending on preparation conditions. The balance of evidence supports the double-occupancy model. Interestingly, the amplitude of the band at 1746 cm[SUP-1] increases with quinone content, reflecting a protonation reaction of acidic groups. In this context, the involvement of glutamates and/or aspartates in the vicinity of the Q[SUB0] site is discussed on the basis of recently determined crystal structures. [ABSTRACT FROM AUTHOR]

Electrostatic interactions are important for protein-protein association. In this study, we examined the electrostatic interactions between two proteins, cytochrome c[sub 2] (cyt c[sub 2]) and the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides, that function in intermolecular electron transfer in photosynthesis. Electrostatic contributions to the binding energy for the cyt c[sub 2]-RC complex were calculated using continuum electrostatic methods based on the recent cocrystal structure [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. Calculated changes in binding energy due to mutations of charged interface residues agreed with experimental results for a protein dielectric constant ∈[sub in] of 10. However, the electrostatic contribution to the binding energy for the complex was close to zero due to unfavorable desolvation energies that compensate for the favorable Coulomb attraction. The electrostatic energy calculated as a function of displacement of the cyt c[sub 2] from the bound position showed a shallow minimum at a position near but displaced from the cocrystal configuration. These results show that although electrostatic steering is present, other short-range interactions must be present to contribute to the binding energy and to determine the structure of the complex. Calculations made to model the experimental data on association rates indicate a solvent-separated transition state for binding in which the cyt c[sub 2] is displaced ∼8 Å above its position in the bound complex. These results are consistent with a two-step model for protein association: electrostatic docking of the cyt c[sub 2] followed by desolvation to form short-range van der Waals contacts for rapid electron transfer. [ABSTRACT FROM AUTHOR]

The use of 5-deazaFAD T491V cytochrome P450 reductase has made it possible to directly measure the rate of electron transfer to microsomal oxyferrous cytochrome (cyt) P450 2B4. In this reductase the FMN moiety can be reduced to the hydroquinone, FMNH[sub 2], while the 5-deazaFAD moiety remains oxidized [Zhang, H., et al. (2003) Biochemistry 42, 6804-6813]. The rate of electron transfer from 5-deazaFAD cyt P450 reductase to oxyferrous cyt P450 was determined by rapidly mixing the ferrous cyt P450-2-electron-reduced 5-deazaFAD T491V reductase complex with oxygen in the presence of substrate. The 5-deazaFAD T491V reductase which can only donate a single electron reduces the oxyferrous cyt P450 and oxidizes to the air-stable semiquinone, with rate constants of 8.4 and 0.37 s[sup -1] at 15 °C. Surprisingly, oxyferrous cyt P450 tums over more slowly with a rate constant of 0.09 s[sup -1], which is the rate of catalysis under steady-state conditions at 15 °C (k[sub cat] = 0.08 s[sup -1]). In contrast, the rate constant for electron transfer from ferrous cyt b[sub 5] to oxyferrous cyt P450 is 10 s[sup -1] with oxyferrous cyt P450 and cyt b[sub 5] simultaneously undergoing spectral changes. Quantitative analyses by LC-MS/MS revealed that the product, norbenzphetamine, was formed with a coupling efficiency of 52% with cyt b[sub 5] and 32% with 5-deazaFAD T491V reductase. Collectively, these results suggest that during catalysis a relatively stable reduced oxyferrous intermediate of cyt P450 is formed in the presence of cyt P450 reductase but not cyt b[sub 5] and that the rate-limiting step in catalysis follows introduction of the second electron. [ABSTRACT FROM AUTHOR]