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The ubihydroquinone:cytochrome c oxidoreductase, or cytochrome bc1, is a central component of respiratory and photosynthetic energy transduction pathways in many organisms. It contributes to the generation of membrane potential and proton gradient used for cellular energy (ATP) production. The three-dimensional structures of cytochrome bc1 show a homodimeric organization of its three catalytic subunits. The unusual architecture revived the issue of whether the monomers operate independently or function cooperatively during the catalytic cycle of the enzyme. In recent years, different genetic approaches allowed the successful production of heterodimeric cytochrome bc1 variants and evidenced the occurrence of intermonomer electron transfer between the monomers of this enzyme. Here we used a version of the "two-plasmid" genetic system, also described in the preceding paper (DOI: 10.1021/bi400560p), to study a new heterodimeric mutant variant of cytochrome bc1. The strain producing this heterodimeric variant sustained photosynthetic growth of Rhodobacter capsulatus and yielded an active heterodimer. Interestingly, kinetic data showed equilibration of electrons among the four b heme cofactors of the heterodimer, via "reverse" intermonomer electron transfer between the bL hemes. Both inactive homodimeric and active heterodimeric cytochrome bc1 variants were purified to homogeneity from the same cells, and purified samples were subjected to mass spectrometry analyses. The data unequivocally supported the idea that the cytochrome b subunits carried the expected mutations and their associated epitope tags. Implications of these findings on our interpretation of light-activated transient cytochrome b and c redox kinetics and the mechanism of function of a dimeric cytochrome bc1 are discussed with respect to the previously proposed heterodimeric Q cycle model. [ABSTRACT FROM AUTHOR]

The candidate tumor suppressor 101F6 protein is a homologue of adrenal chromaffin granule cytochrome b561, which is involved in the electron transfer from cytosolic ascorbate to intravesicular monodehydroascorbate radical. Since the tumor suppressor activity of 101F6 was enhanced in the presence of ascorbate, it was suggested that 101F6 might utilize a similar transmembrane electron transfer reaction. Detailed kinetic analyses were conducted on the detergent-solubilized recombinant human 101F6 for its electron transfer reactions with ascorbate and monodehydroascorbate radical by stopped-flow and pulse radiolysis techniques. The reduction of oxidized 101F6 with ascorbate was found to be independent of pH in contrast to those observed for chromaffin granule and Zea mays cytochromes b561 in which both cytochromes exhibited very slow rates at pH 5.0 but faster at pH 6.0 and 7.0. The absence of the inhibition for the electron acceptance from ascorbate upon the treatment with diethyl pyrocarbonate suggested that 101F6 might not utilize a "concerted proton/electron transfer mechanism". The second-order rate constant for the electron donation from the ascorbate-reduced 101F6 to the pulse-generated monodehydroascorbate radical was found to be 5.0 × 107 M-1 s-1, about 2-fold faster than that of bovine chromaffin granule cytochrome b561 and about five times faster than that of Zea mays cytochrome b561, suggesting that human 101F6 is very effective for regenerating ascorbate from monodehydroascorbate radical in cells. Present observations suggest that 101F6 employs distinct electron transfer mechanisms on both sides of the membranes from those of other members of cytochrome b561 protein family. [ABSTRACT FROM AUTHOR]

We describe photoinitiated electron transfer (ET) from a suite of Zn-substituted myoglobin (Mb) variants to cytochrome b5 (b5). An electrostatic interface redesign strategy has led to the introduction of positive charges into the vicinity of the heme edge through D/E → K charge-reversal mutation combinations at "hot spot" residues (D44, D60, and E85), augmented by the elimination of negative charges from Mb or b5 by neutralization of heme propionates. These variations create an unprecedentedly large range in the product of the ET partners' total charges (-5 < -qMbqb5 < 40). The binding affinity (Ka) increases 1000-fold as -qMbqb5 increases through this range and exhibits a surprisingly simple, exponential dependence on -qMbqb5. This is explained in terms of electrostatic interactions between a "charged reactive patch" (crp) on each partner's surface, defined as a compact region around the heme edge that (i) contains the total protein charge of each variant and (ii) encompasses a major fraction of the "reactive region" (Rr) comprising surface atoms with large matrix elements for electron tunneling to the heme. As -qMbqb5 increases, the complex undergoes a transition from fast to slow-exchange dynamics on the triplet ET time scale, with a correlated progression in the rate constants for intracomplex (ket) and bimolecular (k2) ET. This progression is analyzed by integrating the crp and Rr descriptions of ET into the textbook steady-state treatment of reversible binding between partners that undergo intracomplex ET and found to encompass the full range of behaviors predicted by the model. The generality of this approach is demonstrated by its application to the extensive body of data for the ET complex between the photosynthetic reaction center and cytochrome c2. Deviations from this model also are discussed. [ABSTRACT FROM AUTHOR]

Domain rotation of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c1 redox centers plays a key role in the mechanism of the cyt bc1 complex. Electron transfer within the cyt bc1 complex of Paracoccus denitrificans was studied using a ruthenium dimer to rapidly photo-oxidize cyt c1 within 1 μs and initiate the reaction. In the absence of any added quinol or inhibitor of the bc1 complex at pH 8.0, electron transfer from reduced ISP to cyt c1 was biphasic with rate constants of k1f = 6300 ± 3000 s-1and k1s = 640 ± 300 s-1 and amplitudes of 10 ± 3% and 16 ± 4% of the total amount of cyt c1 photooxidized. Upon addition of any of the Pm type inhibitors MOA-stilbene, myxothiazol, or azoxystrobin to cyt bc1 in the absence of quinol, the total amplitude increased 2-fold, consistent with a decrease in redox potential of the ISP. In addition, the relative amplitude of the fast phase increased significantly, consistent with a change in the dynamics of the ISP domain rotation. In contrast, addition of the Pf type inhibitors JG-144 and famoxadone decreased the rate constant k1f by 5-10-fold and increased the amplitude over 2-fold. Addition of quinol substrate in the absence of inhibitors led to a 2-fold increase in the amplitude of the k1f phase. The effect of QH2 on the kinetics of electron transfer from reduced ISP to cyt c1 was thus similar to that of the Pm inhibitors and very different from that of the Pf inhibitors. The current results indicate that the species occupying the Qo site has a significant conformational influence on the dynamics of the ISP domain rotation. [ABSTRACT FROM AUTHOR]

Cytochrome (cyt) bc1 is a structural dimer with its monomers consisting of the Fe-S protein, cyt b, and cyt c1 subunits. Its three-dimensional architecture depicts it as a symmetrical homodimer, but the mobility of the head domain of the Fe-S protein indicates that the functional enzyme exists in asymmetrical heterodimeric conformations. Here, we report a new genetic system for studying intra- and intermonomer interactions within the cyt bc1 using the facultative phototrophic bacterium Rhodobacter capsulatus. The system involves two different sets of independently expressed cyt bc1 structural genes carried by two plasmids that are coharbored by a cell without its endogenous enzyme. Our results indicate that coexpressed cyt bc1 subunits were matured, assorted, and assembled in vivo into homo- and heterodimeric enzymes that can bear different mutations in each monomer. Using the system, the occurrence of intermonomer electron transfer between the low-potential b hemes of cyt bc1 was probed by choosing mutations that perturb electron transfer at the hydroquinone oxidation (Qo) and quinone reduction (Qi) sites of the enzyme. The data demonstrate that active heterodimeric variants, formed of monomers carrying mutations that abolish only one of the two (Qo or Qi) active sites of each monomer, are produced, and they support photosynthetic growth of R. capsulatus. Detailed analyses of the physicochemical properties of membranes of these mutants, as well as purified homo- and heterodimeric cyt bc1 preparations, demonstrated that efficient and productive electron transfer occurs between the low-potential bL hemes of the monomers in a heterodimeric enzyme. Overall findings are discussed with respect to intra- and intermonomer interactions that take place during the catalytic turnover of cyt bc1. [ABSTRACT FROM AUTHOR]

The green photosynthetic bacterium Chioroflexus auranhiacus, which belongs to the phylum of filamentous anoxygenic phototrophs. does not contain a cytochrome bc or bf type complex which is found in all other known groups of phototrophs. This suggests that a functional replacement exists to link the reaction center photochemistry to cyclic electron transfer as well as respiration. Earlier work identified a potential substitute of the cytochrome be complex, now named alternative complex Ill (ACII I), which has been purified from C. aurantiacus. identified, and characterized. ACIII functions as a menaquinol:auracyanin oxido- reductase in the photosynthetic electron transfer chain, and a related but distinct complex functions in respiratory electron flow to a terminal oxidase. In this work, we focus on elucidating the structure of photosynthetic ACIII. We found that ACIII is an integral membrane protein complex of -3OO kDa that consists of eight subunits of seven different types. Among them, there are four metalloprotein subunits, including a 113 kDa iron-sulfur cluster-containing polypeptide, a 25 kDa penta-heme c-containing subunit, and two 20 kDa monoheme c-containing subunits in the form of a homodimer. A variety of analytical techniques were employed in determining the ACIII substructure, including HPLC combined with ESI-MS, metal analysis, potentiometric titration, and intensity analysis of heme staining SDS-PAGE. A preliminary structural model ofAClil is proposed on the basis of the analytical data and chemical cross-linking in tandem with mass analysis using MALDI-TOF, as well as transmembrane and transit peptide analysis. [ABSTRACT FROM AUTHOR]

Each flavoeytochrome b2 (L-lactate cytochrome c oxidoreductase) subunit consists of an N- terminal cytochrome domain and a C-terminal flavodehydrogenase (FDH) domain. In the enzyme crystal structure, only two heme domains are visible per enzyme tetramer, because of the mobility of the other two heme domains relative to the FDH domains. Evidence was subsequently provided that this mobility also exists in solution. Numerous kinetic studies showed that, during the catalytic cycle, electrons are transferred one by one from the reduced Ilavin to heme b2 in the same subunit. In previous work, we provided evidence that a monoclonal antibody that abolishes flavin to heme electron transfer uses part of the interdomain interface for binding to its antigen, the native heme domain. In this work, we use a number of heme domain side chain substitutions in and around the interface to probe their effect on flavin to heme electron transfer. Using steady-state and pre-steady-state kinetics, as well as redox potential determinations and EPR measurements, we define several hydrophobic interactions and van der Waals contacts that are important for a catalytically competent docking of the heme domain onto the FDH domain. In addition, with several extremely slow mutant enzymes, we propose an isosbestie wavelength between oxidized and reduced heme for specifically following the kinetics of flavosemiquinone formation from two-electron reduced flavin. [ABSTRACT FROM AUTHOR]

Cytochromes b561, a novel class of transmembrane electron transport proteins residing in a large variety of eukaryotic cells, have a number of common structural features including six hydrophobic transmembrane α-helices and two heme ligation sites. We found that recombinant Zea Inays cytochrome b561 obtained by a heterologous expression system using yeast Pichia pastoris cells could utilize the ascorbate/ mondehydroascorbate radical as a physiological electron donor/acceptor. We found further that a concerted proton/electron transfer mechanism might be operative in Z. mays cytochrome b561 as well upon the electron acceptance from ascorbate to the cytosolic heme center. The well-conserved Lys residue in a cytosolic loop was found to have a very important role(s) for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions based on the analyses of three site-specific mutants, K83A, K83E, and K83D. Further, unusual behavior of the K83A mutant in pulse radiolysis experiments indicated that Lys83 might also be responsible for the intramolecular electron transfer to the intravesicular heme. On the other hand, pulse radiolysis experiments on two site-specific mutants, S118A and W122A, for the well-conserved residues in the putative monodehydroascorbate radical binding site showed that their electron transfer activities to the monodehydroascorbate radical were very similar to those of the wild-type protein, indicating that Ser118 and Trp122 do not have major roles for the redox events on the intravesicular side. [ABSTRACT FROM AUTHOR]

The role of short-range hydrogen bond interactions at the interface between electron transfer proteins cytochrome c2 (cyt) and the reaction center (RC) from Rhodobacter sphaeroides was studied by mutation (to Ala) of RC residues Asn M187, Asn M188, and Gin L258 which form interprotein hydrogen bonds to cyt in the cyt-RC complex. The largest decrease in binding constant KA (8-fold) for a single mutation was observed for Asn M187, which forms an intraprotein hydrogen bond to the key residue Tyr L162 in the center of the contact region with a low solvent accessibility. Interaction between Asn M187 and Tyr L162 was also implicated in binding by double mutation of the two residues. The hydrogen bond mutations did not significantly change the second-order rate constant, k2, indicating the mutations did not change the association rate for formation of the cyt-RC complex but increased the dissociation rate. The first-order electron transfer rate, ke, for the cyt-RC complex was reduced by a factor of up to 4 (for Asn M187). The changes in ke were correlated with the changes in binding affinity but were not accompanied by increases in activation energy. We conclude that short-range hydrogen bond interactions contribute to the close packing of residues in the central contact region between the cyt and RC near Asn M187 and Tyr L162. The close packing contributes to fast electron transfer by increasing the rate of electronic coupling and contributes to the binding energy holding the cyt in position for times sufficient for electron transfer to occur. [ABSTRACT FROM AUTHOR]

ABSTRACT: Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c1 and cytochrome c552 was studied using the soluble redox fragments cyt cICF and cyt c552F. A new ruthenium cyt c552F derivative labeled at C23 (Ruzz-23-c552F) was designed to measure rapid electron transfer with cyt cICF in the physiological direction using flash photolysis. The bimolecular rate constant k12 decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by long-range electrostatic interactions between the two proteins. However, a new kinetic phase was detected at an ionic strength of <35 mM with the ruthenium photoexcitation technique in which k12 became very rapid (3 x 109M-1s-1) and nearly independent of ionic strength, suggesting that the reaction became so fast that it was controlled by short-range diffusion along the protein surfaces guided by hydrophobic interactions. These results are consistent with a two-step model for formation of the final encounter complex. No intracomplex electron transfer between Ruz-23-c552F and cICF was observed even at the lowest ionic strength, indicating that the dissociation constant of the complex was >30 μM. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ruz-39-Cc formed a tight 1:1 complex with cyt cICF at ionic strengths of <60 mM with an intracomplex electron transfer rate constant of 50000 s-1. A group of cyt cICF variants in the presumed docking site were generated on the basis of information from the yeast cyt bc1-cyt c cocrystal structure. Kinetic analysis of cyt cICF mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c552F and suggested that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions. [ABSTRACT FROM AUTHOR]

The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of conformations which the protein can adopt in solution and on surfaces of artificial and cell membranes. The structural diversity corresponds to a variety of functions in electron transfer, peroxidase and apoptosis processes. In spite of numerous studies, a comprehensive analysis and comparison of native and non-native states of ferricytochrome c has thus far not been achieved. This results in part from the fact that the influence of solvent conditions (i.e., ionic strength, anion concentration, temperature dependence of pH values) on structure, function and equilibrium thermodynamics has not yet been thoroughly assessed. The current study is a first step in this direction, in that it provides the necessary experimental data to compare different non-native states adopted at high temperature and alkaline pH. To this end, we employed visible electronic circular dichroism (ECD) and absorption spectroscopy to probe structural changes of the heme environment in bovine and horse heart ferricytochrome c as a function of temperature between 278 and 363 K at different neutral and alkaline pH values. A careful selection of buffers enabled us to monitor the partial unfolding of the native state at room temperature while avoiding a change to an alkaline state at high temperatures. We found compelling evidence for the existence of a thermodynamic intermediate of the thermal unfolding/folding process, termed IIIh, which is structurally different from the alkaline states, TV1 and TV2, contrary to current belief. At neutral or slightly acidic pH, IIIh is populated in a temperature region between 320 and 345 K. The unfolded state of the protein becomes populated at higher temperatures. The ECD spectra of the B-bands of bovine and horse heart cytochrome c (pH 7.0) exhibit a pronounced couplet that is maintained below 343 K, before protein unfolding replaces it by a rather strong positive Cotton band. A preliminary vibronic analysis of the B-band profile reveals that the couplet reflects a B-band splitting of 350 cm-1, which is mostly of electronic origin, due to the internal electric field in the heme cavity. Our results suggest that the conformational transition from the native state, III, into a thermally activated intermediate state, IIIh, does not substantially affect the internal electric field and causes only moderate rearrangements of the heme pocket, which involves changes, rather than a rupture, of the Fe3+-M80 linkage. In the unfolded state, as well as in the alkaline states IV and V, the band splitting is practically eliminated, but the positive Cotton effect observed for the B-band suggests that the proximal environment, encompassing H18 and the two cysteine residues 14 and 17, is most likely still intact and covalently bound to the heme chromophore. Both alkaline states IV and V were found to melt via intermediate states. Unfolded states probed at neutral and alkaline pH can be discriminated, owing to the different intensities of the Cotton bands of the respective B-band transitions. Differences between the ECD intensities of the B-bands of the different unfolded states and alkaline states most likely reflect different degrees of openness of the corresponding heme crevice. [ABSTRACT FROM AUTHOR]

The cyanobacterium Synechococcus sp. PCC 7002 carries two genes, petJ1 and petJ2, for proteins related to soluble, cytochrome c6 electron transfer proteins. PetJ1 was purified from the cyanobacterium, and both cytochromes were expressed with heme incorporation in Escherichia coli. The expressed PetJ1 displayed spectral and biochemical properties virtually identical to those of PetJ1 from Synechococcus. PetJ1 is a typical cytochrome c6 but contains an unusual KDGSKSL insertion. PetJ2 isolated from E. coli exhibited absorbance spectra characteristic of cytochromes, although the α, β, and γ bands were red-shifted relative to those of PetJ1. Moreover, the surface electrostatic properties and redox midpoint potential of PetJ2 (pI 9.7; Em,7 148 ± 1.7 mV) differed substantially from those of PetJ1 (pI 3.8; Em,7 = 319 ± 1.6 mV). These data indicate that the PetJ2 cytochrome could not effectively replace PetJ1 as an electron acceptor for the cytochrome bfcomplex in photosynthesis. Phylogenetic comparisons against plant, algal, bacterial, and cyanobacterial genomes revealed two novel and widely distributed clusters of previously uncharacterized, cyanobacterial c6-like cytochromes. PetJ2 belongs to a group that is distinct from both c6 cytochromes and the enigmatic chloroplast c6A cytochromes. We tentatively designate the PetJ2 group as c6C cytochromes and the other new group as c6B cytochromes. Possible functions of these cytochromes are discussed. [ABSTRACT FROM AUTHOR]

Cytochromes bc1/b6f complexes catalyze electron transfer from lipid- to water-soluble carriers in both the respiratory and photosynthetic processes. They contain several common redox cofactors, while a chlorophyll a molecule, the function of which is still enigmatic, is only present in b6f-type complexes. In this work, we describe a mutagenesis approach aimed at characterizing the role of this pigment. Mutants of the binding pocket were constructed to obtain cytochrome (cyt) b6f complexes altered in chlorophyll position and/or stability. On the basis of a combined biochemical and functional analysis, we conclude that the chlorophyll plays a major structural role in the complex. Moreover, the chlorophyll and its binding pocket may also be implicated in the regulation of photosynthetic state transitions, a function that is specific to cyt b6f complexes. [ABSTRACT FROM AUTHOR]

Exchange on the microsecond time scale between the protonated and deprotonated forms of His92 in the copper site of reduced plastocyanin from the cyanobacteria Anabaena variabilis was monitored using 15N NMR relaxation measurements. On the basis of the dependence of the kinetics on pH and phosphate buffer concentration, we propose a two-step model for the protonation of the copper site in agreement with previous crystallographic studies. It is shown that the proton transfer is the rate-limiting step in the reaction at low buffer concentrations, whereas at high buffer concentrations, another step becomes rate-limiting. We suggest that the latter step is a concerted dissociation of His92 from the Cu(I) ion and a 1800 rotation of the imidazole ring, which precede the protonation. The first-order rate constant for the dissociation of His92 from the Cu(I) ion is estimated to be 2.4 × 104 s-1. Also, a cooperative effect of the protonation of the remote His61 on the protonation of His92 and the redox properties of the protein was investigated by substituting His61 with asparagine. The mutation causes a modest change in both the pKa value of His92 and the redox potential of the protein. [ABSTRACT FROM AUTHOR]

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the flavin adenine dinucleotide (FAD/FADH2) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD+/NADH) and donating them in two separate, one-electron-transfer steps to the iron—sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin—NADP+ reductase (FNR), has no other active redox moieties (e.g., sulfhydryl groups) and can exist in three different oxidation states: (i) oxidized quinone, (ii) one-electron reduced semiquinone (stable neutral species (blue) or unstable radical anion (red)), and (iii) two-electron fully reduced hydroquinone. Here, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of equilibria among the redox components in this initial electron-transfer step of the P450 cycle. A spectroelectrochemical technique was used to measure the midpoint oxidation—reduction potential of PdR that had been carefully purified of all residual NAD+, E0' = -369 ± 10 mV at pH 7.6, which is more negative than previously reported and more negative than the pyridine nucleotide NADH/NAD+ (-330 mV). After addition of NAD+, the formation of the oxidized reductase-oxidized pyridine nucleotide complex was followed by the two-electron-transfer redox reaction, PdRox:NAD+ + 2e- → PdRrd:NAD+, when the electrode potential was lowered. The midpoint potential was a hyperbolic function of increasing NAD+ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be E0' = -230 ± 10 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH. This thermodynamic control of electron transfer is a shared mechanistic feature with the adrenodoxin P450 and photosynthetic electron-transfer systems but is different from the kinetic control mechanisms in the microsomal P450 systems where multiple reaction pathways draw on reducing power held by NADPH—cytochrome P450 reductase. The redox measurements were combined with protein fluorescence quenching of NAD+ binding to oxidized PdR to establish that the PdRox:NAD+ complex (KD = 230 μM) is about 5 orders of magnitude weaker than PdRrd:NAD+ binding. These results are integrated with known structural and kinetic information for PdR, as well as for AdR and FNR, in support of a compulsory ordered pathway to describe the electron-transfer processes catalyzed by all three reductases. [ABSTRACT FROM AUTHOR]

The plastocyanin-cytochrome f complex from Nostoc exhibits relevant structural differences when compared with the homologous complexes from other cyanobacteria and plants, with electrostatic and hydrophobic interactions being differently involved in each case. Here, five negatively charged residues of a recombinant form of cytochromef from Nostoc have been replaced with either neutral or positively charged residues, and the effects of mutations on the kinetics of electron transfer to wild-type and mutant forms of plastocyanin have been measured by laser flash absorption spectroscopy. Cytochromef mutants with some negative charges replaced with neutral residues exhibit an apparent electron transfer rate constant with wild-type plastocyanin similar to or slightly higher than that of the wild-type species, whereas the mutants with negative charges replaced with positive residues exhibit a significantly lower reactivity. Taken together, these results indicate that the effects of neutralizing residues at the electrostatically charged patch of cytochrome f are smaller than those previously observed for mutants of plastocyanin, thus suggesting that it is the copper protein which determines the specificity of the electrostatic interaction with the heme protein. Moreover, cross reactions between mutants of both proteins reveal the presence of some short-range specific electrostatic interactions. Our findings also make evident the fact that in Nostoc the main contribution to the electrostatic nature of the complex is provided by the small domain of cytochrome f. [ABSTRACT FROM AUTHOR]

Typically, c hemes are bound to the protein through two thioether bonds to cysteines and two axial ligands to the heme iron. In high-potential class I c-type cytochromes, these axial ligands are commonly His-Met. A change in this methionine axial ligand is often correlated with a dramatic drop in the heme redox potential and loss of function. Here we describe a bacterial cytochrome c with an unusual tolerance to the alternations in the heme ligation pattern. Substitution of the heme ligating methionine (M185) in cytochrome c1 of the Rhodobacter sphaeroides cytochrome bc1 complex with Lys and Leu lowers the redox midpoint potential but not enough to prevent physiologically competent electron transfer in these fully functional variants. Only when Met-185 is replaced with His is the drop in the redox potential sufficiently large to cause cytochrome bc1 electron transfer chain failure. Functional mutants preserve the structural integrity of the heme crevice: only the nonfunctional His variant allows carbon monoxide to bind to reduced heme, indicating a significant opening of the heme environment. This range of cytochrome c1 ligand mutants exposes both the relative resilience to sixth axial ligand change and the ultimate thermodynamic limits of operation of the cofactor chains in cytochrome bc1. [ABSTRACT FROM AUTHOR]

Cytochrome P450cam (P450cam) is the terminal monooxygenase in a three-component camphor-hydroxylating system from Pseudomonas putida. The reaction cycle requires two distinct electron transfer (ET) processes from the [2Fe-2S] containing putidaredoxin (Pdx) to P450cam. Even though the mechanism of interaction and ET between the two proteins has been under investigation for over 30 years, the second reductive step and the effector role of Pdx are not fully understood. We utilized mutagenesis, kinetic, and computer modeling approaches to better understand differences between the two Pdx-to-P450cam ET events. Our results indicate that interacting residues and the ET pathways in the complexes formed between reduced Pdx (Pdxг) and the ferric and ferrous dioxygen-bound forms of P450cam (oxy-P450cam) are different. Pdx Asp38 and Trpl06 were found to be key players in both reductive steps. Compared to the wild-type Pdx, the D38A, WI06A, and Δ106 mutants exhibited considerably higher Kd values for ferric P450cam and retained ca. 20% of the first electron transferring ability. In contrast, the binding affinity of the mutants for oxy-P450cam was not substantially altered while the second ET rates were < 1%. On the basis of the kinetic and modeling data we conclude that (i) P450cam¯ Pdx interaction is highly specific in part because it is guided/controlled by the redox state of both partners; (ii) there are alternative ET routes from Pdxг to ferric P450cam and a unique pathway to oxy-P450cam involving Asp38; (iii) Pdx Trp106 is a key structural element that couples the second ET event to product formation possibly via its ‘push’ effect on the heme-binding loop. [ABSTRACT FROM AUTHOR]

Internal electron transfer (ET) to heme a3 during anaerobic reduction of oxidized bovine heart cytochrome c oxidase (CcO) was studied under conditions where heme a and CuA were fully reduced by excess hexaamineruthenium. The data show that ET to heme a3 is controlled by the state of ionization of a single protolytic residue with a pKa of 6.5 ± 0.2. On the basis of the view that ET to the catalytic site is limited by coupled proton transfer, this pKa was attributed to Glu60 which is located at the entrance of the proton-conducting K channel on the matrix side of CcO. It is proposed that Glu60 controls proton entry into the channel. However, even with this channel open, there is the second factor that regulates ET, and this is ascribed to the rate of proton diffusion in the channel. In addition, it is concluded that proton transfer in the K channel is reversibly inhibited by the detergent Triton X- 100. It is also found that the rate of ET to heme a3 in the as-isolated resting enzyme and in CcO "activated" by reaction of fully reduced enzyme with O2 is the same, implying that the catalytic sites of these two forms of oxidized enzyme are essentially identical. [ABSTRACT FROM AUTHOR]

The reaction between cytochrome f and plastocyanin is a central feature of the photosynthetic electron-transport system of all oxygenic organisms. We have studied the reaction in solution to understand how the very weak binding between the two proteins from Phormidium laminosum can nevertheless lead to fast rates of electron transfer. In a previous publication [Schlarb-Ridley, B. G., et al. (2003) Biochemistry 42,4057-4063], we suggested that the reaction is diffusion-controlled because of a strong effect of viscosity of the medium. The effects of viscosity and temperature have now been examined in detail. High molecular mass viscogens (Ficoll 70 and Dextran 70), which might mimic in vivo conditions, had little effect up to a relative viscosity of 4. Low molecular mass viscogens (ethane diol, glycerol, and sucrose) strongly decreased the bimolecular rate constant (k2) over a similar viscosity range. The effects correlated well with the viscosities of the solutions of the three reagents but not with their dielectric constants or molalities. A power law dependence of k2 on viscosity suggested that k2 depends on two viscosity-sensitive reactions in series, while the reverse reactions are little affected by viscosity. The results were incompatible with diffusion control of the overall reaction. Determination of the effect of temperature on k2 gave an activation enthalpy, Δwhich is also incompatible with diffusion control. The results were interpreted in terms of a model in which the stable form of the protein-protein complex requires further thermal activation to be competent for electron transfer. [ABSTRACT FROM AUTHOR]

The highly efficient electron-transfer chain in photosynthesis demonstrates a remarkable variation among organisms in the type of interactions between the soluble electron-transfer protein plastocyanin and it partner cytochrome f. The complex from the cyanobacterium Nostoc sp. PCC 7119 was studied using nuclear magnetic resonance spectroscopy and compared to that of the cyanobacterium Phormidium laminosum. In both systems, the main site of interaction on plastocyanin is the hydrophobic patch. However, the interaction in the Nostoc complex is highly dependent on electrostatics, contrary to that of Phormidium, resulting in a binding constant that is an order of magnitude larger at low ionic strength for the Nostoc complex. Studies of the mixed complexes show that these differences in interactions are mainly attributable to the surface properties of the plastocyanins. [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]

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 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 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 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]

The camphor hydroxylase cytochrome P450[sub cam] (CYP101) catalyzes the 5-exo hydroxylation of camphor in the first step of camphor catabolism by Pseudomonas putida. CYP101 forms a specific electron transfer complex with its physiological reductant, the Cys[sub 4]Fe[sub 2]S[sub 2] ferredoxin putidaredoxin (Pdx). Pdx, along with other proteins and small molecules, has also been shown to be an effector for turnover by CYP101. Multidimensional nuclear magnetic resonance (NMR) techniques have been used to make extensive sequential ¹H, [sup 15]N, and [sup 13]C resonance assignments in CYP101 that permit a more complete characterization of the complex formed by CYP101 and Pdx. NMR-detected perturbations in CYP101 upon Pdx binding encompass regions of the CYP101 remote from the putative Pdx binding site, including in particular a region of the CYP101 molecule that has been implicated in substrate access to the active site via dynamical processes. A model for effector activity is proposed in which the primary role of the effector is to prevent uncoupling (formation of reduced oxo species without formation of hydroxycamphor) by enforcing conformations of CYP101 that prevent loss of substrate and/or intermediates prior to turnover. A secondary role could also be to enforce conformations that permit efficient proton transfer into the active site for coupled proton/electron transfer. [ABSTRACT FROM AUTHOR]

Cytochrome f and plastocyanin from the cyanobacterium Phormidium laminosum react an order of magnitude faster than their counterparts from chloroplasts when long-range electrostatic interactions have been screened out by high salt concentration [Schlarb-Ridley, B. G., et al. (2002) Biochemistry 41, 3279-3285]. To investigate the relative contributions of the reaction partners to these differences, the reactions of turnip cytochrome f with P. laminosum plastocyanin and P. laminosum cytochrome f with pea plastocyanin were examined. Exchanging one of the plant reaction partners with the corresponding cyanobacterial protein nearly abolished electron transfer at low ionic strength but increased the rate at high ionic strength. This increase was larger for P. laminosum cytochrome f than for P. laminosum plastocyanin. To identify molecular features of P. laminosum cytochromefthat contribute to the increase, the effect of mutations in the N-terminal heme-shielding peptide on the reaction with P. laminosum plastocyanin was determined. Phenylalanine-3 was converted to valine and tryptophan-4 to phenylalanine or leucine. The mutations lowered the rate constant at 0.1 M ionic strength by factors of 0.71 for F4V, 0.42 for W4F, and 0.63 for W4L while introducing little change in the shape of the ionic strength dependence curve. When the N-terminal tetrapeptide (sequence YPFW) was converted into that found in the chloroplast of Chlamydomonas reinhardtii (YPVF), the reaction was slowed further (factor of 0.26). The N-terminal heme-shielding peptide was found to be responsible for 75% of the kinetic differences between cytochrome f from chloroplasts and the cyanobacterium when electrostatic interactions were eliminated. [ABSTRACT FROM AUTHOR]

Describes the mutational analyses of the photosynthetic reaction center-bound triheme cytochrome subunit. Physiological role of proteins for photosynthetic electron transfer; Conformation of chromosomal insertion; Effects of residual soluble cytochromes on closed chromatophores.

Focuses on the electron transfer from c heme to d[sub 1] heme in bacterial cytochrome cd[sub 1] nitrite reductase. Occurrence of the transfer over the same distances; Relevance of Pseudomonas aeruginosa enzymes; Rate constant for the transfer.

Reports that ascorbate inhibits the carbethoxylation of two histidyl and one tyrosyl residues indispensable for the transmembrane electron transfer reaction of cytochrome b[sub 561]. Enhancement of the O-carbethoxylation of one tyrosul residue; Presence of ascorbate during the treatment with diethyl pyrocarbonate.

Examines the effect of the protein matrix on the reorganization energy and electronic coupling of cytochrome. Comparison with the behavior of porphyrin complexes; Effect of the orientation of heme relative to the electrode; Implications for the understanding of long-range interheme protein electron transfer.

Examines the side-chain interaction between the redox proteins cytochrome f and plastocyanin in the photosynthetic electron-transfer chain. Effect of mutations on the structure of plastocyanin-cytochrome f complex; Nature of the interactions in the hydrophobic and acidic patches; Site of largest chemical-shift changes.

Studies electron transfer between the Rieske iron-sulfur protein (Fe2S2) and cytochrome c1 using the ruthenium dimer, Ru2D, to either photoreduce or photooxidize cytochrome c1 within 1 microsecond. Development of a ruthenium photoexcitation method to measure the rate constant for a key step in the cytochrome bc1 complex electron transfer.

Examines the uphill electron transfer (ET) in the tetraheme cytochrome subunit of the photosynthetic reaction center from Rhodopseudomonas viridis. High redox potentiality of the heme3 among the hemes in the cyt unit; Kinetics of the ET from the tetraheme cytochrome to the special pair; Details on calculations of activation energy and ET rates among hemes.

Investigates the electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli using pulse radiolysis. Reaction of the air-oxidized enyzme with hydrogen peroxide; Reduction of the hemes by the N-methylnicotinamide radical; Influence of the oxidation and litigation states of heme d on the heme b reduction.

Presents a comparative study of the photoinduced electron-transfer reaction between the triplet state of zinc cytochrome c and cupriplastocyanin. Materials and methods of the study; Association of the proteins with zinc cytochrome c; Effect of mutation in tyrosine 83 on the rate constant K...; Effects of temperature and of plastocyanin mutation on the rate constant K...

Presents information pertaining to the electron and proton transfers within the cytochrome (cyt) ... complex, while examining the effect which N,N'-dicyclohexylcarbodiimide (DCCD) can have in living cells of the green algae Chlorella sorokiniana under anaerobic conditions. Capabilities of DCCD; Association of the formation of a transmembrane potential and the transfer of proton with electron-transfer reactions; Inclusion of four electron carriers.

Presents a hypothetical model for electron transfer complex between cytochrome c3 and the flavodoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris. Generation, expression, and characterization of Flavodoxin mutants; Electron transfer from reduced cytochrome c3 to oxidized Flavodoxin.

Studies electron transfer within cytochrome cd1 from Thiosphaera pantotropha with the use of pulse radiolysis technique. Reduction of the heme centers in the nitrite reductase; Evidence of intramolecular electron transfer from the c-heme to the d1-heme.

Discusses the effects of enzyme inhibitor decyl-aurachin D and reversed electron transfer on cytochrome bd from Azotobacter vinelandii. Nature of the cytochrome bd complex; Significance of the interaction of decyl-aurachin D with the oxidase; Structure and mechanism of the oxidase.

Studies the cyclic electron transfer in Heliobacillus mobilis involving a menaquinol-oxidizing cytochrome bc complex and an RCI-type reaction center. Investigation of flash-induced absorption changes arising from b-type hemes; Redox potential dependence of reduction and oxidation reactions of cytochrome b-hemes.

Studies the electron transfer in cytochromes D. vulgaris Hildenborough and D. desulfuricans Norway by thin layer spectroelectrochemistry. Characterization of the intermediate state; Resonance Raman spectra; Evidence for conformational change triggered by heme IV reduction.

Expands the physical characterization of the electron transfer (ET) dynamics of cytochromes by using time-resolved and transient resonance Raman spectroscopy. Examination of the vibrational structure of the heme active sites during and immediately subsequent to photoinitiated ET in two different ruthenated cytochromes; Observed structural responses to ET.

Studies the effect of temperature on the kinetics of the reaction induced by a laser pulse both in purified reaction centers solubilized in detergent suspensions and in reconstituted proteoliposomes restoring the native lipid environment of the protein. Purification of reaction centers; Reconstitution of proteoliposomes; Interaction of cytochrome C2.

Investigates the role of tyrosine L162 in docking and electron transfer from the cyt heme to P+ in Rhodobacter sphaeroides. Bacterial strains and growth; Recombinant DNA techniques; Protein purification techniques; Spectroscopy; Flash kinetic measurements.

Introduces a method to study electron transfer that involves photoexcitation of a covalently attached tris(bipyrimidine)ruthenium complex. Electron-transfer reaction between cytochrome b5 and cytochrome c; Preparation; Flash photolysis experiments.