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We identified a novel human cDNA encoding a mitochondrial protein, MTP18 (mitochondrial protein, 18 kDa) as a transcriptional downstream target of phosphatidylinositol (PI) 3-kinase signaling. We demonstrate that MTP18 mRNA as well as protein expression is dependent on PI 3-kinase activity. Confocal microscopy and biochemical fractlonation revealed a mitochondrial localization of MTP18. Loss-of-function analysis employing antisense molecules revealed that MTP18 is essential for cell viability in PC-3 and HaCaT cells. We show that knockdown of MTP18 protein level results in a cytochrome c release from mitochondria and consequently leads to apoptosis. In addition, HaCaT cells with reduced levels of MTP18 become more sensitive to apoptotic stimuli. This effect is accompanied by dramatic subcellular alterations. Reduction of MTP18 impairs mitochondrial morphology resulting in the formation of a highly interconnected mitochondrial reticulum in COS-7 cells. Conversely, overexpression of MTP18 induces a punctuate morphology of mitochondria suggesting also a functional role of MTP18 in maintaining the mitochondrial integrity. Hence, our data indicate an unexpected connection of PI 3-kinase signaling, apoptosis and the morphology of mammalian mitochondria. [ABSTRACT FROM AUTHOR]

We have investigated the role of the mitochondrial pathway during cell death following serum and nerve growth factor (NGF)/dibutyryl cyclic AMP (Bt2cAMP) withdrawal in undifferentiated or NGF/Bt2cAMP-differentiated PC12 cells, respectively. Holocytochrome c, Smac/DIABLO, and Omi/HtrA2 are released rapidly following trophic factor deprivation in PC12 cells. Bcl-2 and Akt inhibited this release. The protection, however, persisted longer in differentiated PC12 cells. In differ. entiated, but not undifferentiated cells, Bcl-2 and Akt also inhibited apoptosis downstream of holocytochrome c release. Thus, undifferentiated PC12 cells showed marked sensitivity to induction of apoptosis by micro. injected cytochrome c even in the presence of NGF, Bcl-2, or Akt. In contrast, in differentiated cells these factors suppressed cell death. Consistent with these observations, in vitro processing of procaspase 9 in response to cytochrome c was observed in extracts from undifferentiated but not differentiated cells expressing Akt or Bcl-2. Endogenous caspase 9 was cleaved during cell death, whereas dominant negative caspase 9 inhibired cell death. The results from determining the role of inhibitors of apoptosis (IAPs) suggest that acquisition of inhibition by IAPs is part of the differentiation program. Ubiquitin-ΔN-AVPI Smac/DIABLO induced cell death in differentiated cells only. c-IAP-2 is unregulated in differentiated cells, whereas X-linked 1AP levels decreased in these cells coincident with cell death. Moreover, expressing X-linked IAP rendered undifferentiated cells resistant to microinjected cytochrome c. Overall, the inhibitory regulation, of cell death at the level of release of mitochondrial apoptogenic factors and at post-mitochondrial activation of caspase 9 observed in differentiated PC12 cells, is reduced or absent in the undifferentiated counterparts. [ABSTRACT FROM AUTHOR]

Previous studies on certain altered holo-isocytochromes c revealed a ρ--dependent degradation (RDD) phenotype, in which certain altered holo-iso-1-cytochromes c are at normal or nearly normal levels in ρ+ strains, but are at low levels or absent in ρ- strains, although wild-type holo-iso-1-cytochrome c is present at normal levels in both ρ+ and related ρ- strains. The diminished levels of altered holo-iso-1-cytochrome c are due to the rapid degradation that is carried out by a novel proteolytic pathway in the IMS of mitochondria. SUE1, a nuclear gene that encodes a mitochondrial protein, was identified with a genetic screen for mutants that diminish RDD. The levels of RDD and certain other types of altered holo-iso-1-cytochrome c were elevated in ρ- sue1 strains. Also, ρ+ sue1 strains containing certain altered holo-iso-1-cytochromes c grew better on non-fermentable carbon sources than the corresponding ρ+ SUE1 strains. These results indicate that Sue1p may play an important role in the degradation of abnormal holo-iso-1-cytochrome c in the mitochondria. [ABSTRACT FROM AUTHOR]

Heme-oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation, has powerful anti-oxidant properties related to the production of the reactive oxygen species scavenger bilirubin. However, some data suggest that HO-1 could also inhibit the cellular production of reactive oxygen species. Therefore, we investigated whether the anti.oxidant properties of HO-1 could be mediated by modulation of the activity and/or expression of the heme-containing NAD(P)H oxidase, the main source of the superoxide anion (O2&˙macr;) in phagocytic cells. Increasing HO-1 expression in RAW 264.7 macrophages effectively decreased NAD(P)H oxidase activity and expression of gp91phox, its heme-containing catalytic component, because of deficient protein maturation and increased degradation. Loading cells with heme reversed the decrease in O2&˙macr; production and gp91phox expression induced by HO-1 overexpression. Similar results were obtained in vivo in rat alveolar macrophages after pharmacological modulation of HO-1 expression or activity. These results show that a decrease in heme content due to HO-1 activation limits heme availability for maturation of the gp91phox subunit and assembly of the functional NAD(P)H oxidase. This study provides a new mechanism to explain HO-1 anti-oxidant properties. [ABSTRACT FROM AUTHOR]

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47phox, p67phox, p40phox, and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b55s (cyt b558) with different amounts of FAD. A remarkably high activity, over 100 μmol/s/μmol heme, was obtained in the oxidase with purified cyt b558, ternary complex (p47–p67-p40phox), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b558, the dissociation constant Kd of FAD in cyt b558 of reconstituted oxidase was estimated as nearly I nM. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b55s increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b558 recovered electron flow from NADPH to heme. From both Kd values of FAD and the amount of incorporated FAD in cyt b558 of reconstituted oxidase, in combination with spectra, we propose the model in which the Kd values of FAD in cyt b558 is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase. [ABSTRACT FROM AUTHOR]

The cytochrome bound to the photosynthetic reaction center of Rhodovulum sulfidophilum presents two unusual characteristics with respect to the well characterized tetraheme cytochromes. This cytochrome contains only three hemes because it lacks the peptide motif CXXCH, which binds the most distal fourth heme. In addition, we show that the sixth axial ligand of the third heme is a cysteine (Cys-148) instead of the usual methionine ligand. This ligand exchange results in a very low midpoint potential (−160 ± 10 mV). The influence of the unusual cysteine ligand on the midpoint potential of this distal heme was further investigated by site-directed mutagenesis. The midpoint potential of this heme is upshifted to +310 mV when cysteine 148 is replaced by methionine, in agreement with the typical redox properties of a His/Met coordinated heme. Because of the large increase in the midpoint potential of the distal heme in the mutant, both the native and modified high potential hemes are photooxidized at a redox poise where only the former is photooxidizable in the wild type. The relative orientation of the three hemes, determined by EPR measurements, is shown different from tetraheme cytochromes. The evolutionary basis of the concomitant loss of the fourth heme and the down-conversion of the third heme is discussed in light of phylogenetic relationships of the Rhodovulum species tri-heme cytochromes to other reaction center-associated tetraheme cytochromes. [ABSTRACT FROM AUTHOR]

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evo idence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination. [ABSTRACT FROM AUTHOR]

The fungus Gibberella fujikuroi is used for the commercial production of glbberellins (GAs), which it produces in very large quantities. Four of the seven GA biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH-cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD, FMN, and NADPH binding motifs, cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA3, GA4, and GA7 production, but low levels of non-hydroxylated C20-GAs (GA15 and GA24) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monooxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAS. Northern blot analysis revealed co-regulated expression of the cpr. Gf gene and the GA biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism. [ABSTRACT FROM AUTHOR]

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunoflu. orescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytoehrome c in inducing nuclear apoptosis. [ABSTRACT FROM AUTHOR]

In yeast, Tim50 along with Tim23 regulate translocation of presequence-containing proteins across the mitochondrial inner membrane. Here, we describe the identification and characterization of a novel human mitochondrial inner membrane protein homologous to the yeast Tim50. We demonstrate that human Tim50 possesses phosphatase activity and is present in a complex with human Tim23. Down-regulation of human Tim50 expression by RNA interference increases the sensitivity of human cell lines to death stimuli by accelerating the release of cytochrome c from the mitochondria. Furthermore, injection of Tim50-specific morpholino antisense oligonucleotides during early zebrafish embryonic development causes neurodegeneration, dysmorphic hearts, and reduced motility as a result of increased cell death. These observations indicate that loss of Tim50 in vertebrates causes mitochondrial membrane permeabilization and dysfunction followed by cytoplasmic release of cytochrome c along with other mitochondrial inducers of cell death. Thus Tim50 is important for both mitochondrial function and early neuronal development. [ABSTRACT FROM AUTHOR]

The mammalian CYP1A1, CYP1A2, and CYP1B1 genes (encoding cytochromes P450 1A1, 1A2, and 1B1, respectively) are regulated by the aromatic hydrocarbon receptor (AHR). The CYP1 enzymes are responsible for both metabolically activating and detoxifying numerous polycyclic aromatic hydrocarbons (PAHs) and aromatic amines present in combustion products. Many substrates for CYP1 enzymes are AHR ligands. Differences in AHR affinity between inbred mouse strains reflect variations in CYP1 inducibility and clearly have been shown to be associated with differences in risk of toxicity or cancer caused by PAHs and arylamines. Variability in the human AHR affinity exists, but differences in human risk of toxicity or cancer related to AHR activation remain unproven. Mouse lines having one or another of the Cyp1 genes disrupted have shown paradoxical effects; in the test tube or in cell culture these enzymes show metabolic activation of PAHs or arylamines, whereas in the intact animal these enzymes are sometimes more important in the role of detoxification than metabolic potentiation. Intact animal data contradict pharmaceutical company policies that routinely test drugs under development; if a candidate drug shows CYP1 inducibility, further testing is generally discontinued for fear of possible toxic or carcinogenic effects. In the future, use of ‘humanized’ mouse lines, containing a human AHR or CYP1 allele in place of the orthologous mouse gene, is one likely approach to show that the AHR and the CYP1 enzymes in human behave similarly to that in mouse. [ABSTRACT FROM AUTHOR]

Post-translational maturation of cytochromes c involves the covalent attachment of heme to the Cys-XxxXxx-Cys-His motif of the apo-cytochrome. For this process, the two cysteines of the motif must be in the reduced state. In bacteria, this is achieved by dedicated, membrane-bound thiol-disulfide oxidoreductases with a high reducing power, which are essential components of cytochrome c maturation systems and are also linked to cellular disulfide-bond formation machineries. Here we report high-resolution structures of oxidized and reduced states of a soluble, functional domain of one such oxidoreductase, ResA, from Bacillus subtilis. The structures elucidate the structural basis of the protein's high reducing power and reveal the largest redox-coupled conformational changes observed to date in any thioredoxin-like protein. These redox-coupled changes alter the protein surface and illustrate how the redox state of ResA predetermines to which substrate it binds. Furthermore, a polar cavity, present only in the reduced state, may confer specificity to recognize apo-cytochrome c. The described features of ResA are likely to be general for bacterial cytochrome c maturation systems. [ABSTRACT FROM AUTHOR]

On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp627 is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochrome c6 to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002) J. Biol. Chem. 277, 6573-6581). To examine the function of PsaA-Trp651 in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation of Chlamydomonas reinhardtii. The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt) c6 by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp651 to Ser had an even larger impact on the dissociation constant. The KO value increased another 2-fold when the values obtained for the interaction and electron transfer between cyt c6 and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an intermolecular electron transfer complex, resulting in a Ko value of about 554 µM that is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp651 is, such as PsaB-Trp627, crucial for high affinity binding of pc and cyt c6 to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp651 and PsaB-Trp627 confers a differential selectivity in binding of beth donors to PSI. [ABSTRACT FROM AUTHOR]

Cytochrome c release is a central step in the apoptosis induced by many death stimuli. Bcl-2 plays a critical role in controlling this step. In this study, we investigated the upstream mechanism of cytochrome c release induced by ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a recently discovered small molecule inhibitor of Bcl-2. HA14-1 was found to induce cytochrome c release from the mitochondria of intact cells but not from isolated mitochondria. Cytochrome c release from isolated mitochondria requires the presence of both HA14-1 and exogenous Ca2+. This suggests that both mitochondrial and extramitochondrial signals are important. In intact cells, treatment with HA14-1 caused Ca2+ spike, change in mitochondrial membrane potential (ΔΨm) transition, Bax translocation, and reactive oxygen species (ROS) generation prior to cytochrome c release. Pretreatment with either EGTA acetoxymethyl ester or vitamin E resuited in a significant decrease in cytochrome c release and cell death induced by HA14-1. Furthermore pretreatment with RU-360, an inhibitor of the mitochondrial Ca2+ uniporter, or with EGTA acetoxymethyl ester, but not with vitamin E, prevented the HA14-1-induced ΔΨm transition and Bax translocation. This suggests that ROS generation is an event that occurs after the ΔΨm transition and Bax translocation. Together these data demonstrate that the Ca2+ spike, mitochondrial Bcl-2 presensitization, and subsequent ΔΨm transition, Bax translocation, and ROS generation are important upstream signals for cytochrome c release upon HA14-1 stimulation. The involvement of endoplasmic reticulum and mitochondrial signals suggests both organelles are crucial for HA14-1-induced apoptosis. [ABSTRACT FROM AUTHOR]

Conversion of Hydrogenobacter thermophilus cytochrome c552 into a b-type cytochrome by mutagenesis of both heme-binding cysteines to alanines significantly reduces the stability of the protein (Tomlinson, E. J., and Ferguson, S. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5156-5160). To understand the effects of this change on the structure and dynamics of the protein, heteronuclear 15N-edited NMR techniques have been used to characterize this b-type variant. The backbone 15N, ¹HN, and ¹Hα, and ¹Hβ resonances of the protein have been assigned. Analysis of ³JHNα coupling constants, nuclear Overhauser enhancement intensities, and chemical shift index data demonstrates that the four α-helices present in the wild-type protein are retained in the btype variant. Comparison of the chemical shifts for the b-type and wild-type proteins indicates that the tertiary structures of the two proteins are closely similar. Some subtle differences are, however, observed for residues in the N-terminal region and in the vicinity of the hemebinding pocket. Hydrogen exchange studies show that there are 25 backbone amide protons that exchange very slowly in the b-type variant and confirm that the fluctuations within the b-type protein are of a similar extent to those in the wild-type protein. These data demonstrate the notable retention of the native secondary structure and tertiary fold despite the absence of covalent linkages between the heme group and the protein. [ABSTRACT FROM AUTHOR]

In the crystal structure of chicken sulfite oxidase, the residue Tyr322 (Tyr343 in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6-10. In steady-state assays of Y343F sulrite oxidase using cytochrome c as the electron acceptor, kcat was somewhat impaired (∼34% wild-type activity at pH 8.5), whereas the Kmsulfite showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, kredheme changed very little over the entire pH range, with a significant increase in Kdsulfiteat high pH. The kredheme of the Y343F variant was significantly impaired across the entire pH range, and unlike the wildtype protein, both kredheme and Kdsulfite were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (kredMo = 972 s-1 at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr343 residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase. [ABSTRACT FROM AUTHOR]

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc1 complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c1 reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is ∼200 mV and in a bc1 complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c1 reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc1 complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc1 complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two bH hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinolcytochrome c reductase activity of the bc1 complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc1 dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc1 complex dimer. [ABSTRACT FROM AUTHOR]

During apoptosis, cytochrome c is released from mitochondria into the cytosol, where it participates in caspase activation. Various and often conflicting mechanisms have been proposed to account for the increased permeability of the mitochondrial outer membrane that is responsible for this process. The voltage-dependent anion channel (VDAC) is the major permeability pathway for metabolites in the mitochondrial outer membrane and therefore is a very attractive candidate for cytochrome c translocation. Here, we report that properties of VDAC channels reconstituted into planar phospholipid membranes are unaffected by addition of the pro-apoptotic protein Bax under a variety of conditions. Contrary to other reports (Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483-487; Shimizu, S., Ide, T., Yanagida, T., and Tsujimoto, Y. (2000) J. Biol. Chem. 275, 12321-12325; Shimizu, S., Konishi, A., Kodama, T., and Tsujimoto, Y. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 3100-3105), we found no electrophysiologically detectable interaction between VDAC channels isolated from mammalian mitochondria and either monomeric or oligomeric forms of Bax. We conclude that Bax does not induce cytochrome c release by acting on VDAC. In contrast to Bax, another pro-apoptotic protein (Bid) proteolytically cleaved with caspase-8 affected the voltage gating of VDAC by inducing channel closure. We speculate that by decreasing the probability of VDAC opening, Bid reduces metabolite exchange between mitochondria and the cytosol, leading to mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Here the cytochrome c peroxidase (CcP) from Nitrosomonas europaea is examined using the technique of catalytic protein film voltammetry. Submonolayers of the bacterial diheme enzyme at a pyrolytic graphite edge electrode give catalytic, reductive signals in the presence of the substrate hydrogen peroxide. The resulting waveshapes indicate that CcP is bound non-covalently in a highly active configuration. The native enzyme has been shown to possess two heme groups of low and high potential (L and H, -260 and +450 mV versus hydrogen, respectively), and here we find that the catalytic waves of the N. europaea enzyme have a midpoint potential of > 500 mV and a shape that corresponds to a 1-electron process. The signals increase in magnitude with hydrogen peroxide concentration, revealing Michaelis-Menten kinetics and Km = 55 µM. The midpoint potentials shift with substrate concentration, indicating the electrochemically active species observed in our data corresponds to a catalytic species. The potentials also shift with respect to pH, and the pH dependence is interpreted in terms of a two pKa model for proton binding. Together the data show that the electrochemistry of the N. europaea cytochrome c peroxidase is unlike other peroxidases studied to date, including other bacterial enzymes. This is discussed in terms of a catalytic model for the N. europaea enzyme and compared with other cytochrome c peroxidases. [ABSTRACT FROM AUTHOR]

We report a multifrequency (9.6-, 94-, 190-, and 285GHz) EPR study of a freeze-quenched intermediate obtained from reaction of substrate-free cytochrome P450cam (CYP101) and its Y96F and Y96F/Y75F mutants with peroxy acids. It is generally assumed that in such a shunt reaction an intermediate [Fe(IV)=O, porphyrin-πcation radical] is formed, which should be identical to the species in the natural reaction cycle. However, for the wild type as well as for the mutant proteins, a porphyrin-π-cation radical is not detectable within 8 ms. Instead, EPR signals corresponding to tyrosine radicals are obtained for the wild type and the Y96F mutant. Replacement of both Tyr-96 and Tyr-75 by phenylalanine leads to the disappearance of the tyrosine EPR signals. EPR studies at 285 GHz on freeze-quenched wild type and Y96F samples reveal g tensor components for the radical (stretched gx values from 2.0078 to 2.0064, gy = 2.0043, and gz = 2.0022), which are fingerprints for tyrosine radicals in a heterogeneous polar environment. The measurements at 94 GHz using a fundamental mode microwave resonator setup confirm the 285-GHz study. From the simulation of the hyperfine structure in the 94-GHz EPR spectra the signals have been assigned to Tyr-96 in the wild type and to Tyr-75 in the Y96F mutant. We suggest that a transiently formed Fe(IV)=O porphyrin-π-cation radical intermediate in P450cam is reduced by intramolecular electron transfer from these tyrosines within 8 ms. [ABSTRACT FROM AUTHOR]

Ilicicolin H is an antibiotic isolated from the ‘imperfect’ fungus Cylindrocladium iliciola strain MFC-870. Ilicicolin inhibits mitochondrial respiration by inhibiting the cytochrome bc1 complex. In order to identify the site of ilicicolin action within the bc1 complex we have characterized the effects of ilicicolin on the cytochrome bc1 complex of Saccharomyces cerevisiae. Ilicicolin inhibits ubiquinol-cytochrome c reductase activity of the yeast bc1 complex with an IC50 of 3–5 nM, while 200–250 nM ilicicolin was required to obtain comparable inhibition of the bovine bc1 complex. Ilicicolin blocks oxidation-reduction of cytochrome b through center N of the bc1 complex and promotes oxidant-induced reduction of cytochrome b but has no effects on oxidation of ubiquinol through center P. These results indicate that ilicicolin binds to the Qn site of the bc1 complex. Ilicicolin induces a blue shift in the absorption spectrum of ferro-cytochrome b, and titration of the spectral shift indicates binding of one inhibitor molecule per Qn site. The effects of ilicicolin on electron transfer reactions in the bc1 complex are similar to those of antimycin, another inhibitor that binds to the Qn site of the bc1 complex. However, because the two inhibitors have different effects on the absorption spectrum of cytochrome b, they differ in their mode of binding to the Qn site. [ABSTRACT FROM AUTHOR]

In cyanobacteria, cytochrome c6 and plastocyanin are able to replace each other as redox carriers in the photosynthetic and respiratory electron transport chains with the synthesis of one or another protein being regulated by the copper concentration in the culture medium. However, the presence of a third unidentified electron carrier has been suggested. To address this point, we have constructed two deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803, each variant lacking either the pete or petJ gene, which respectively codes for the copper or heme protein. The photoautotrophic and heterotrophic growth rate of the two mutants in copper-free and copper-supplemented medium as well as their photosystem I reduction kinetics in vivo were compared with those of wild-type cells. The two mutant strains grow at equivalent rates and show similar in vivo photosystem I reduction kinetics as wildtype cells when cultured in media that allow the expression of just one of the two electron donor proteins, but their ability to grow and reduce photosystem I is much lower when neither cytochrome c6 nor plastocyanin is expressed. These findings indicate that the normal functioning of the cyanobacterial photosynthetic and respiratory chains obligatorily depends on the presence of either cytochrome c6 or plastocyanin. [ABSTRACT FROM AUTHOR]

Atovaquone is a substituted hydroxynaphthoquinone that is widely used to prevent and clear Plasmodium falciparum malaria and Pneumocystis jirovecii pneumonia. Atovaquone inhibits respiration in target organisms by specifically binding to the ubiquinol oxidation site at center P of the cytochrome bc[sub 1] complex. The failure of atovaquone treatment and mortality of patients with malaria and P. jirovecii pneumonia has been linked to the appearance of mutations in the cytochrome b gene. To better understand the molecular basis of atovaquone resistance, we have introduced seven of the mutations from atovaquone-resistant P. jirovecii into the cytochrome b gene of Saccharomyces cerevisiae and thus obtained cytochrome bc[sub 1] complexes resistant to inhibition by atovaquone. In these enzymes, the IC[sub 50] for atovaquone increases from 25 nM for the enzyme from wild-type yeast to >500 nM for some of the mutated enzymes. Modeling of the changes in cytochrome b structure and atovaquone binding with the mutated bc[sub 1] complexes provides the first quantitative explanation for the molecular basis of atovaquone resistance. [ABSTRACT FROM AUTHOR]

Provides evidence that cardiolipin Is not required for bax-mediated cytochrome c release from yeast mitochondria. Synthetic liposomes; Liquid chromatography-mass spectrometry; Absence of the yeast CL species.

The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carties the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heine b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI. [ABSTRACT FROM AUTHOR]

Retinoids are potent regulators of cell proliferation, cell differentiation, and morphogenesis and are important therapeutic agents in oncology and dermatology. The gene regulatory activity of endogenous retinoids is effected primarily by retinoic acid isomers (all-trans and 9-cis) that are synthesized from retinaldehyde precursors in a broad range of tissues and act as ligands for nuclear retinoic acid receptors. The catabolism of alltrans-retinoic acid (atRA) is an important mechanism of controlling RA levels in cell and tissues. We have previously identified two cytochrome P450s, P450RAI-1 and P450RAI-2 (herein named CYP26A1 and CYP26B1), which were shown to be responsible for catabolism of atRA both in the embryo and the adult. In this report, we describe the identification, molecular cloning, and substrate characterization of a third member of the CYP26 family, named CYP26C1. Transiently transfected cells expressing CYP26C1 convert atRA to polar water-soluble metabolites similar to those generated by CYP26A1 and -B1. Competition studies with all-trans, 13-cis, and 9-cis isomers of retinoic acid demonstrated that atRA was the preferred substrate for CYP26C1. Although CYP26C1 shares extensive sequence similarity with CYP26A1 and CYP26B1, its catalytic activity appears distinct from those of other CYP26 family members. Specifically, CYP26C1 can also recognize and metabolize 9-cis-RA and is much less sensitive than the other CYP26 family members to the inhibitory effects of ketoconazole. CYP26C1 is not widely expressed in the adult but is inducible by RA in HPK1a, transformed human keratinocyte cell lines. This third CYP26 member may play a specific role in catabolizing both all-trans and 9-cis isomers of RA. [ABSTRACT FROM AUTHOR]

The photosynthetic reaction center (RC) from Rhodopseudomonas viridis contains four cytochrome c hemes. They establish the initial part of the electron transfer (ET) chain through the RC. Despite their chemical identity, their midpoint potentials cover an interval of 440 mV. The individual heme midpoint potentials determine the ET kinetics and are therefore tuned by specific interactions with the protein environment. Here, we use an electrostatic approach based on the solution of the linearized Poisson-Boltzmann equation to evaluate the determinants of individual heme redox potentials. Our calculated redox potentials agree within 25 meV with the experimentally measured values. The heme redox potentials are mainly governed by solvent accessibility of the hemes and propionic acids, by neutralization of the negative charges at the propionates through either protonation or formation of salt bridges, by interactions with other hemes, and to a lesser extent, with other titratable protein side chains. In contrast to earlier computations on this system, we used quantum chemically derived atomic charges, considered an equilibrium-distributed protonation pattern, and accounted for interdependencies of site-site interactions. We provide values for the working potentials of all hemes as a function of the solution redox potential, which are crucial for calculations of ET rates. We identify residues whose site-directed mutation might significantly influence ET processes in the cytochrome c part of the RC. Redox potentials measured on a previously generated mutant could be reproduced by calculations based on a model structure of the mutant generated from the wild type RC. [ABSTRACT FROM AUTHOR]

The mitochondrial permeability transition (MPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the MPT, we used diethylraaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory complex III (cytochrome bc[sub 1]), because 1) stigmatellin, a Q[sub o] site inhibitor, blocked ROS production and prevented the MPT and cell death and 2) cytochrome bc[sub 1] activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q[sub 10] analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent MPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the MPT and cell death, whereas Ub0 did not. Since we also found that dub did not inhibit cytochrome bc[sub 1] activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc[sub 1]. These results indicate that dub, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component. [ABSTRACT FROM AUTHOR]

Gene inactivation studies point to the involvement of OxyC in catalyzing the last oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. Presently, the substrate and exact timing of the OxyC reaction are unknown. The substrate might be the bicyclic heptapeptide or a thioester derivative bound to a protein carrier domain. OxyC from the vancomycin producer Amycolatopsis orientalis was produced in Escherichia coli and crystallized, and its structure was determined to 1.9 Å resolution. OxyC gave UV-visible spectra characteristic of a P450-1ike hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. The addition of vancomycin aglycone to OxyC produced type I changes to the UV spectrum. OxyC exhibits the typical P450-fold, with the Cys ligand loop containing the signature sequence FGHGXHXCLG and Cys-356 being the proximal axial thiolate ligand of the heme iron. The observation of a water molecule bound to the heme iron is consistent with the UV-visible spectra of OxyC indicating a low spin heme. A polyethylene glycol molecule occupying the active site might mimic the bicyclic heptapeptide substrate. Analysis of the structure of Oxy-proteins and other P450s indicates regions that might be involved in binding of the redox partner and possibly the protein carrier domain. [ABSTRACT FROM AUTHOR]

This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid. A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E. coli. Its progressive induction in the presence of 5-aminolevulinic acid-supplemented medium was accompanied by an accumulation of a greater than 100-fold higher amount of uroporphyrin I in the periplasm relative to cells lacking CYP105DI. Expression of a cytoplasm-resident engineered CYP105D1 at a comparative level to the secreted form was far less effective in promoting porphyrin accumulation in the periplasm. Expression at a 10-fold molar excess over the exported CYP105D1 of another periplasmically exported hemoprotein, the globular core of cytochrome b[sub 5], did not substitute the role of the periplasmically localized CYP105D1 in promoting porphyrin production. This, therefore, eliminated the possibility that uroporphyrin accumulation is merely a result of increased hemoprotein synthesis. Moreover, in the strain that secreted CYP105D1, uroporphyrin production was considerably reduced by azole-based P450 inhibitors. Production of both holo-CYP105D1 and uroporphyrin was dependent upon 5-aminolevulinic acid, except that at higher concentrations this resulted in a decrease in uroporphyrin. This study suggests that the exported CYP105D1 oxidatively catalyzes periplasmic conversion of uroporphyrinogen I to uroporphyrin I in E. coli. The findings have significant implications in the ontogenesis of human uroporphyria-related diseases. [ABSTRACT FROM AUTHOR]

The 2.3-kb mRNA that codes for cytochrome P-450c27 (CYP27) has an unexpectedly long 5'-untranslated region (UTR) that holds six AUGs, leading to several upstream open reading frames (uORFs). The initiation of translation from the seventh AUG forms a putative 55kDa precursor, which is processed in mitochondria to form a 52-kDa mature protein. The first three AUGs form fully overlapping uORF1, uORF2, and uORF3 that are in-frame with the seventh AUG and next two form fully overlapping uORF4 and uORF5 that are out-of-frame with the seventh AUG. Although not recognized by the scanning ribosomes under normal conditions, the sixth in-frame AUG forms a putative 57-kDa extension of the main open reading frame. The purpose of this study was to identify the elements in the 5'-UTR that direct CYP27 mRNA translation exclusively from the seventh AUG. Expression of 5' deletion mutants in COS cells reveal that the intact 5'-UTR not only directs the initiation of translation from the seventh AUG but also acts as a negative regulator. A 2-kb deletion mutant that lacks uORF1 initiates translation equally from the sixth and the seventh AUGs, forming both 57- and 55-kDa precursor proteins with a 2-fold increase in rate of translation. However, induction in translation does not affect the levels of the mature 52-kDa form in mitochondria but causes accumulation of the precursor form in cytosol not seen in COS cells transfected with wild-type cDNA. Mutation of the stop codon that terminates uORF1 completely shifts the initiation of translation from the seventh to the first AUG, forming a 67-kDa precursor that is processed into a 52-kDa mature protein in mitochondria. Confirmation of the bicistronic nature of CYP27 mRNA by epitope mapping of uORF1 suggests that translation of CYP27 mRNA from the seventh AUG is directed and regulated by uORF1 expression. [ABSTRACT FROM AUTHOR]

Presents a study where a cDNA library prepared from hepatic mRNA of sterol 27-hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionary conserved microsomal cytochrome P450 with vitamin D 25-hydroxylase activity. Transcriptional activation of the vitamin D receptor; Thin layer chromatography and radioimmunoassays on the secosteroid product of CYP2R1.

Determines the crystal structure of the oxidized and reduced forms of the nine-heme cytochrome at pH 7.5 and performs theoretical calculations of the binding equilibrium of protons and electrons in these structures. Observation that the reduction process induced conformation changes in several residues; Evidence that the N- and C-terminal domains function in an independent manner.

The cytochrome P450 CYP2B19 is a keratinocyte-specific arachidonic acid epoxygenase expressed in the granular cell layer of mouse epidermis. In cultured keratinocytes, CYP2B19 mRNAs are up-regulated coordinately with those of profilaggrin, another granular cellspecific marker. We investigated effects of the CYP2B19 metabolites 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on keratinocyte transglutaminase activities and cornified cell envelope formation. Keratinocytes were differentiated in vitro in the presence of biotinylated cadaverine. Transglutaminases cross-linked this substrate into endogenous proteins in situ; an enzymelinked immunosorbent assay was used to quantify the biotinylated proteins. Exogenously added or endogenously formed 14,15-EET increased transglutaminase cross-linking activities in cultured human and mouse epidermal keratinocytes in a modified in situ assay. Transglutaminase activities increased ∼8-fold (p ≤ 0.02 versus mock control) in human keratinocytes transduced with adenovirus particles expressing a 14S,15REET epoxygenase (P450 BM3v). The physiological transglutaminase substrate involucrin was preferentially biotinylated in situ, determined by immunoblotting and mass spectrometry. P450 BM3v-induced transglutaminase activation was associated with increased 14,15-EET formation (p = 0.002) and spontaneous cell cornification (p ≤ 0.001). Preferential involucrin biotinylation and the increased cornified cell envelope formation provided evidence that transglutaminases mediated the P450 BM3v-induced cross-linking activities. These results support a physiological role for 14,15-EET epoxygenases in regulating epidermal cornification, and they have important implications for epidermal barrier functions in vivo. [ABSTRACT FROM AUTHOR]

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heine with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met[sup 65] was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 Å resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N[sup δ1] and N[sup δ2] nitrogen atoms, rather than the typical N[sup ∈2]/N[sup ∈2] coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N[sup δ1]/N[sup ∈2]-coordinated protoporphyrin IX iron. [ABSTRACT FROM AUTHOR]

Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc[sub 1] complex. We have examined the interaction of atovaquone with the bc[sub 1] complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc[sub 1] complex competitively with apparent K[sub i] = 9 nM, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc[sub 1] complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc[sub 1] complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc[sub 1] complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc[sub 1] complex (K[sub i] = 80 nM) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (K[sub i] = 100 nM) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc[sub 1] complex and explain the differential inhibition of the fungal versus mammalian enzymes. [ABSTRACT FROM AUTHOR]

Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc[sub 1] complex catalysis. Binding of the competitive inhibitor 5-n-heptyl6-hydroxy-4,7-dioxobenzothiazole to the Q[sub o] site of the cytochrome bc[sub 1] complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pK[sub a] = 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His[sub 181] and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr[sup 279]. The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His[sup 181] with a pK[sub a] of 7.5. Glu[sup 272], a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme b[sub L] propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Q[sub o] site. [ABSTRACT FROM AUTHOR]

Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. In this study, we investigated the involvement of the forkhead box, class O (FOXO) family of transcription factors and their downstream target p27[sup Kip1] in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12EET induced a time- and dose-dependent decrease in p27[sup Kip1] protein expression, whereas p21[sup Cip1] was not significantly affected. This effect on p27[sup Kip1] protein was associated with decreased mRNA levels as well as p27[sup Kip1] promoter activity, 11,12-EET also stimulated the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominantnegative or an "Akt-resistant"/constitutively active FOXO3a mutant reversed the 11,12-EET-induced downregulation of p27[sup Kip1], whereas transfection of a constitutive active Akt decreased p27[sup Kip1] expression independently of the presence or absence of 11,12-EET. To determine whether these effects are involved in EETinduced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicited endothelial cell proliferation and this effect was inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. Reducing FOXO expression using RNA interference, on the other hand, attenuated p27[sup Kip1] expression and stimulated endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt.dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27[sup Kip1]. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis MR-1 has the metabolic capacity to grow anaerobically using Fe(III) as a terminal electron acceptor. Growth under these conditions resuits in the de novo synthesis of a number of periplasmic c-type cytochromes, many of which are multiheme in nature and are thought to be involved in the Fe(III) respiratory process. To begin a biochemical study of these complex cytochromes, the mtrA gene that encodes an approximate 32-kDa periplasmic decaheme cytochrome has been heterologously expressed in Escherichia coli. Co-expression of mtrA with a plasmid that contains cytochrome c maturation genes leads to the assembly of a correctly targeted holoprotein, which covalently binds ten hemes. The recombinant MtrA protein has been characterized by magnetic circular dichroism, which shows that all ten hemes have bishistidine axial ligation. EPR spectroscopy detected only eight of these hemes, all of which are low spin and provides evidence for a spin-coupled pair of hemes in the oxidized state. Redox titrations of MtrA have been cartied out with optical- and EPR-monitored methods, and the hemes are shown to reduce over the potential range -100 to -400 mV. In intact cells of E. coli, MtrA is shown to obtain electrons from the host electron transport chain and pass these onto host oxidoreductases or a range of soluble Fe(III) species. This demonstrates the promiscuous nature of this decaheme cytochrome and its potential to serve as a soluble Fe(III) reductase in intact cells. [ABSTRACT FROM AUTHOR]

Discusses the in vitro formation of heme-attached AXXXCh and CXXXAH variants of hydrogenobacter thermophilus from the corresponding apocytochrome and heme. Reaction of apocytochromes with the heme derivatives 2-vinyldeuteroheme and 4-vinyldeuteroheme; Comparison of visible spectra of non-covalent and covalent complexes of the wild-type hydrogenobacter cytochrome; Spontaneous formation of a single thioether bond.

Discusses a study to determine the viral factor potentially involved in viral persistence in chronic hepatitis C virus leading to liver cancer. Interaction of NS2 with the liver-specific pro-apoptotic CIDE-B protein; Results of two-hybrid screen on human liver cDNA library; Colocalization studies and coprecipitation experiments on human endogenous CIDE-B; Release of cytochrome from mitochondria.

Discusses a study on a constructed ResA-deficient Bacillus subtilis strain found to lack c-type cytochromes. Restoration of Cytochrome c synthesis in the mutant; In vivo studies of ResA; Analysis of a soluble form of the protein.

Discusses the physiological role of two calcium sites present in Desulfovibrio desulfuricans cytochrome c nitriate reductase (NrfA). Structure solution by molecular replacement; NrfA from D. desulfuricans ATCC 27774; Determination of crystal structures for the catalytic subunit NrfA.

Highlights the achievements of David Keilin in biological science. Brief profile of Keilin; Story of how Keilin came upon cytochrome when studying hemoglobin in the horse intestinal parasite Gastrophilus intestinalis; Expansion of Keilin's respiratory chain; Function of the respiratory chain; Oxidative phosphorylation and topography of the respiratory chain; Mechanism of electron transfer.

Demonstrates that the trypanosoma brucei cytochrome c[sub1] protein is imported along a non-conservative pathway similar using a mitochondrial protein import assay. Need by the pathway for an external adenosine triphosphate and an external protein receptor; Translocation across the outer mitochondrial membrane; Succession of a heme attachment and membrane insertion into the inner membrane.

Studies oxygen gas migration in the putative oxygen gas channel of cytochrome ba[sub3] and its effects to the properties of the binuclear heme from Thermus thermophilous. Use of the Fourier transform infrared spectra; Absence of any changes on the frequencies of the C-O stretching modes of heme; Indication of the rate of decay of the transient Cu[subb][sup1+]-CO complex.

Examines the inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase. Changes in the in vivo metabolism of pentobarbital and acetaminophen; Normal development of CPR-null mice; Suggestion that the role for hepatic P450 is to protect mammals from their environment.

Reports on the crystal structure and kinetic properties of Paracoccus pantotrophus cytochrome nitrite reductase with the heme active site ligand tyrosine 25 replaced by serine. Expression of the protein; Electrospray mass spectrometry; Structure of the Y25S mutant; Visible absorption spectra; Steady-state kinetic assays.

Discusses the effect of famoxadone on photoinduced electron transfer between the iron-sulfur center and cytochrome c[sub 1] in the cytochrome bc[sub 1]. Rate constant for electron transfer in the forward direction; Characteristic of the reverse electron transfer from cyt c[sub 1] to 2Fe-2S; Electron transfer in Rhodobacter sphaeroides.

Cites the substrate recognition of fatty acid hydroxylation by cytochrome P450 from bacillus subtilis. Molecular mass of bacillus subtilis; Use of hydrogen peroxide as oxidant; Presence of hydrophobic residues in the enzyme.