A study of the electron transfer properties of the heme undecapeptide from cytochrome c by 1H nmr spectroscopy

Nuclear magnetic resonance (nmr) spectroscopy has been used to investigate the heme undecapeptide from cytochrome c. Assignments of resonances to specific residues have been made based on spin decoupling, redox titration, and the pH and temperature dependence of resonance lines. An outline structure is presented based on the assignments, secondary shift data, and the x-ray crystal structure of cytochrome c. An equation is derived to relate the width of an nmr line during a redox titration to the percentage of each oxidation state. Using this equation the self-exchange rate constant for electron transfer for the heme peptide is 1.3 x 10(7) M-1 sec-1 at 330 degrees K. Discussion of the self-exchange rate constants of cytochrome c, cytochrome c3, and cytochrome c551 is related to this constant for the heme undecapeptide.     

Proton nuclear magnetic resonance characterization of the oxidized intermediates of cytochrome c peroxidase

Oxidation of cytochrome c peroxidase with hydrogen peroxide to form the initial oxidized intermediate, cytochrome c peroxidase compound I, drastically alters the proton hyperfine nmr spectrum. In contrast to studies of horseradish peroxidase, where the spectrum of horseradish peroxidase compound I is similar to that of the native protein, cytochrome c peroxidase compound I exhibits only broad resonances near 17 and 30 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate. No unique resonances attributable to cytochrome c peroxidase compound II could be identified. These results define the molecular conditions for which resolved hyperfine resonances of the iron(IV) states of heme proteins may be observed when the data presented here are compared with the data from horseradish peroxidase. Oxidation of cytochrome c peroxidase while it is complexed to ferricytochrome c reveals that the heme resonances of cytochrome c are not influenced by the oxidation state of cytochrome c peroxidase.     

Movement of the iron-sulfur subunit beyond the ef loop of cytochrome b is required for multiple turnovers of the bc1 complex but not for single turnover Qo site catalysis.

Recent kinetics experiments using mutants of the bc(1) complex (ubihydroquinone-cytochrome c oxidoreductase) iron-sulfur subunit with modified hinge regions have revealed the crucial role played by the large scale movement of its [2Fe-2S] cluster domain during the activity of this enzyme. In particular, one of these mutants (+1Ala) with an insertion of one alanine residue in the hinge region is partially deficient in performing this movement. We found that this defect can be overcome by the appearance of a second mutation substituting the leucine at position 286 in the ef loop of cytochrome b with a phenylalanine. Detailed studies of these mutants and their derivatives revealed that the ef loop acts as a barrier that needs to be crossed for multiple turnovers of the enzyme but not for a single turnover ubihydroquinone oxidation site catalysis. These findings indicate that the movement of the iron-sulfur subunit is composed of two discrete parts: a "micro-movement" at the cytochrome b interface, during which the [2Fe-2S] cluster interacts with ubihydroquinone oxidation site occupants and catalyzes ubihydroquinone oxidation, and a "macro-movement," during which the cluster domain swings away from cytochrome b interface, crosses the ef loop, and reaches a position close to cytochrome c(1) heme, to which it ultimately transfers an electron.     

Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme

This paper contributes to the characterization of partial steps of electron and proton transfer in mitochondrial cytochrome c oxidase with respect to their membrane arrangement and involvement in energy-linked protonmotive activity. It is shown that delta psi controls electron flow from cytochrome c to heme a is consistent with the view that the latter center is buried in the membrane in a central position. The pressure exerted by delta psi on oxidation of heme alpha 3 by O2 indicates also that this center is buried in the membrane at some distance from the inner side and is consistent with observations showing that protons consumed in the reduction of O2 to H2O derive from the inner space. Electron flow from heme alpha to heme alpha 3 is shown to be specifically controlled by delta pH and in particular by the pH of the inner phase. Analysis of the effect of DCCD treatment of oxidase vesicles reveals that concentrations of this reagent which result in selective modification of subunit III (Prochaska et al., 1981) produce inhibition of redox-linked proton release. Higher concentrations of DCCD which result also in modification of subunits II and IV (Prochaska et al., 1981) cause inhibition of the pH-dependent electron-transfer step from heme alpha to heme alpha 3.     

Kinetics of flavin semiquinone reduction of the components of the cytochrome c-cytochrome b5 complex

The kinetics of flavin semiquinone reduction of the components of the 1:1 complex formed by cytochrome c with either cytochrome b5 or a derivative of cytochrome b5 in which the heme propionates are esterified (DME-cytochrome b5) have been studied. The rate constant for the reduction of horse heart cytochrome c by the electrostatically neutral lumiflavin semiquinone (LfH) is unaffected by complexation with native cytochrome b5 at pH 7. However, complex formation with DME-cytochrome b5 (pH 7) decreases by 35% the rate constant for cytochrome c reduction by LfH. At pH 8, complex formation with native cytochrome b5 decreases the rate constant for cytochrome c reduction by LfH markedly, whereas the rate constant for cytochrome c reduction, either unbound or in the complex formed with DME-cytochrome b5, is increased 2-fold relative to pH 7. These results indicate that the accessibility of the cytochrome c heme is not the same in the complexes formed with the two cytochrome b5 derivatives and that the docking geometry of the complex formed by the two native cytochromes is pH dependent. Binding of horse heart and tuna cytochromes c to native and DME-cytochromes b5 decreases the rate constants for reduction of cytochrome c by the negatively charged flavin mononucleotide semiquinone (FMNH) by approximately 30% and approximately 40%, respectively. This finding is attributed to substantial neutralization of the positive electrostatic potential surface of cytochrome c that occurs when it binds to either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy tranduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the co-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata.

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.     

Haem ligands of the ferricytochrome c of Ustilago sphaerogena

The mammalian-type cytochrome c of the basidiomycete Ustilago sphaerogena contains in a single polypeptide chain of 107 residues, two histidine residues located at positions 18 and 33, and one methionine residue situated at position 80 (Bitar et al., 1972). The reaction of Ustilago ferricytochrome c with bromoacetate at neutral pH resulted in the modification of histidine-33, but not of histidine-18 or of the invariant methionine residue. The activities of Ustilago cytochrome c with mitochondrial cytochrome c oxidase and with NADH-cytochrome c reductase were unaltered by the modification. The equilibrium constants for the formation of low-spin complexes of the ferrihaem octapeptide of horse cytochrome c (residues 14-21, including the haem bound covalently to cysteines 14 and 17) with imidazole, N(2)-acetylhistidine and monocarboxymethyl derivatives of N(2)-acetylhistidine were determined spectrophotometrically. Alkylation of the imidazole side-chain group of N(2)-acetylhistidine resulted in a marked decrease in its ability to form low-spin ferrihaem complexes. These results indicate that in Ustilago ferricytochrome c in solution histidine-33 is not involved in the central co-ordination complex. Since side-chain groups of residues other than histidine and methionine do not appear to be involved in the central complexes of other mammalian-type cytochromes c (Hettinger & Harbury, 1964, 1965; Myer & Harbury, 1965) it is likely that in Ustilago ferricytochrome c in solution at neutral pH, the side-chain groups of histidine-18 and methionine-80 are involved in the central co-ordination complex. The latter is stable over the pH range 2.6-8.4.     

Characterization of steady-state activities of cytochrome c oxidase at alkaline pH: mimicking the effect of K-channel mutations in the bovine enzyme

The cytochrome c oxidase activity of the bovine heart enzyme decreases substantially at alkaline pH, from 650 s(-1) at pH 7.0 to less than 10 s(-1) at pH 9.75. In contrast, the cytochrome c peroxidase activity of the enzyme shows little or no pH dependence (30-50 s(-1)) at pH values greater than 8.5. Under the conditions employed, it is demonstrated that the dramatic decrease in oxidase activity at pH 9.75 is fully reversible and not due to a major alkaline-induced conformational change in the enzyme. Furthermore, the Km values for cytochrome c interaction with the enzyme were also not significantly different at pH 7.8 and pH 9.75, suggesting that the pH dependence of the activity is not due to an altered interaction with cytochrome c at alkaline pH. However, at alkaline pH, the steady-state reduction level of the hemes increased, consistent with a slower rate of electron transfer from heme a to heme a3 at alkaline pH. Since it is well established that the rate of electron transfer from heme a to heme a3 is proton-coupled, it is reasonable to postulate that at alkaline pH, proton uptake becomes rate-limiting. The fact that this is not observed when hydrogen peroxide is used as a substrate in place of O2 suggests that the rate-limiting step is proton uptake via the K-channel associated with the reduction of the heme a3/CuB center prior to the reaction with O2. This step is not required for the reaction with H2O2, as shown previously in the examination of mutants of bacterial oxidases in which the K-channel was blocked. It is concluded that at pH values near 10, the delivery of protons via the K-channel becomes the rate-limiting step in the catalytic cycle with O2, so that the behavior of the bovine enzyme resembles that of the K-channel mutants in the bacterial enzymes.     

Resonance Raman spectroscopy indicates a lysine as the sixth iron ligand in cytochrome f

The resonance Raman spectrum of turnip cytochrome f is similar to that of other c-type cytochromes with the exception of a single band at 1532 cm-1 which is shifted to lower frequency relative to its position (1542-1545 cm-1) in other c-type cytochromes. Comparison of the frequency of this band with that in alkylated cytochrome c at high pH suggests that the sixth heme iron ligand in cytochrome f is a deprotonated lysine amino group rather than a methionine sulfur. Comparison of the amino-acid sequences of cytochromes f and c1 suggests lysine-145 as a likely candidate for the sixth heme iron ligand in cytochrome f.     

MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association.

The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely.The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit.The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur.     

Kinetics of dithionite reduction of the heme nonapeptide of cytochrome c

The kinetics of dithionite reduction of the oxidized heme nonapeptide fragment of horse heart cytochrome c have been measured as a function of ionic strength at pH 7 and pH 9 by the stopped-flow technique. Dithionite concentration dependences indicate that the radical anion monomer, SO2-., is the active reductant. The pH 7 ionic strength dependence suggests that the heme peptide is reacting as a negatively charged molecule (its overall charge is calculated to be -1). Comparison of these results with the known rate of dithionite reduction of cytochrome c indicates that the heme nonapeptide has substantially greater inherent reactivity than cytochrome c, perhaps due to the greater accessibility of the heme.     

Physicochemical characterization of the alkaline denaturation of cytochrome c peroxidase

The alkaline denaturation of cytochrome c peroxidase and apocytochrome c peroxidase was investigated by analytical ultracentrifugation, gel-filtration chromatography, and circular dichroism. The results indicate that both cytochrome c peroxidase and the apoenzyme undergo extensive structural modifications upon exposure to alkaline pH, including dimer formation. The midpoint of the transition for dimer formation in the native enzyme occurs at pH 9.6 +/- 0.1, while loss of tertiary and secondary structure occurs with transition midpoints at pH 10.3 +/- 0.1 and pH 11.3 +/- 0.1, respectively. Studies performed in the presence of dithiothreitol and with carboxymethylated cytochrome c peroxidase indicate that dimer formation occurs via a disulfide crosslink involving the single cysteine residue in the enzyme. Denaturation of cytochrome c peroxidase in the presence of guanidine hydrochloride gave results similar to those obtained for the alkaline denaturation.     

Kinetic characterization of cytochrome c oxidase from Bacillus subtilis

Bacillus subtilis aa3-type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins. The kinetic properties of the enzyme are influenced by ionic strength. The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant. An increase of Vmax is observed when the enzyme is incorporated in artificial membranes. Negatively charged phospholipids allow high turnover rates of the aa3-type oxidase. The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax. These effects can be explained by disturbances of enzyme-substrate interactions and are not related to changes in the aggregation state of the enzyme. The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH. The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton-permeability measurements of the membranes indicate that both components of the proton-motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state.     

Characterization of the reaction between ferrocytochrome c and cytochrome c oxidase.

The reaction between cytochrome c oxidase and ferrocytochrome c has been investigated by the stopped-flow method. It has been found that only one electron acceptor, a heme group, in the oxidase is rapidly reduced by cytochrome c. The presence of N3- does not affect the reduction of the acceptor, which supports the hypothesis that this is identical with cytochrome a. The results are consistent with the existence of a simple equilibrium between cytochrome a and cytochrome c: c-2 + a-3+ in equilibrium c-3+ + a-2+ with an equilibrium constant corresponding to an oxidation-reduction potential of cytochrome a 30 mV higher than that for cytochrome c at pH 7.4. The oxidation-reduction potential of the a-3+ /a-2+ couple, 285 mV (based on a potential of 255 mV for cytochrome c), and the optical properties of the reduced form indicate that it is identical with neither of the reduced hemes seen in potentiometric titrations. The oxidase species resulting from the rapid reduction of cytochrome a by cytochrome c is proposed to represent a metastable intermediate state which, under anaerobic conditions, eventually is transformed into a more stable state characterized by a reduced high-potential heme.     

Some properties of mammalian cardiac cytochrome c1.

Investigations into the nature of the axial heme ligands, the strength of the heme crevice, the reactivity with cyanide, and the ascorbate reducibility of cytochrome c1 were performed to explore structure-function relationships of cytochrome c1. The existence of an absorbance band at 690 nm, which was quenched by raising the pH with a pK of 9.2 corresponding to a low spin-low transition, suggested that a methionine residue probably functioned as one of the axial heme iron ligands in this cytochrome. Spectral titrations of cytochrome c1 in the low pH range showed a markedly elevated pK for the low spin-high spin transition relative to cytochrome c. Denaturation studies with urea, the absence of any reaction with cyanide, and the evidence from other lines would appear to indicate that the heme group of cytochrome c1 was reduced by ascorbate at approximately 5% of the rate of reduction of cytochrome c but this rate dramatically increased with increasing pH concomitant with the disappearance of the 690 nm absorbance band. Circular dichroic spectra substantiated that elevated pH produced conformational changes localized to the heme crevice and probably also the regions containing aromatic residues. The enhanced rate of ascorbate reduction was perhaps a consequence of the increased accessibility of the heme iron to ascorbate. Major unfolding of the protein in 8 M urea, however, completely abolished the ascorbate reducibility of cytochrome c1. The buried nature of the heme group of cytochrome c1 would probably preclude transfer of an electron from cytochrome c1 to cytochrome c through a direct Fe-Fe or a heme-heme interaction. This poses an important question concerning the mechanism of this electron transfer between these two cytochromes not only in mitochondria but also in solution.     

The role of cytochrome c4 in bacterial respiration. Cellular location and selective removal from membranes.

The cellular location of cytochrome c4 in Pseudomonas stutzeri and Azotobacter vinelandii was investigated by the production of spheroplasts. Soluble cytochrome c4 was found to be located in the periplasm in both organisms. The remaining cytochrome c4 was membrane-bound. The orientation of this membrane-bound cytochrome c4 fraction was investigated by proteolysis of the cytochrome on intact spheroplasts. In P. stutzeri, 78% of the membrane-bound cytochrome c4 could be proteolysed, whilst 82% of the spheroplasts remained intact, suggesting that the membrane-bound cytochrome c4 is on the periplasmic face of the membrane in this organism. Cytochrome c4 was not susceptible to proteolysis on A. vinelandii spheroplasts, in spite of being digestible in the purified state. Cytochrome c5 was shown to have a similar cellular distribution to cytochrome c4. Selective removal of cytochrome c4 from membranes of P. stutzeri was accomplished by the use of sodium iodide and propan-2-ol, with the retention of most of the ascorbate-TMPD (NNN'N'-tetramethylbenzene-1,4-diamine) oxidase activity associated with the membrane. Sodium iodide removed most of the cytochrome c4 from A. vinelandii membranes with retention of 62% of the ascorbate-TMPD oxidase activity. Cytochrome c4 could be returned to the washed membranes, but with no recovery of this enzyme activity. We conclude that cytochrome c4 is not involved in the ascorbate-TMPD oxidase activity associated with the membranes of these two organisms.     

The electron transfer system of Pseudomonas aeruginosa: a study of the pH-dependent transitions between redox forms of azurin and cytochrome c551

Fast reaction kinetic experiments on the electron transfer reaction between azurin and cytochrome c₅₅₁ isolated from Pseudomonas aeruginosa confirmed the existence of two redox forms of reduced azurin previously reported. The pH dependence of the amplitudes of the relaxation processes observed in temperature jump experiments indicate that these two redox forms are in pH dependent equilibrium. The pH independence of the overall equilibrium constant indicates that redox active and inactive forms of cytochrome c₅₅₁ may also exist. Evidence that reduced cytochrome c₅₅₁ undergoes a pH transition is given by optical spectrophotometry. The nature of the transition is discussed in the context of recent nmr studies and in terms of the Marcus theory of electron transfer. The metabolic consequences of these transitions are also discussed.     

Enzymatic properties of ubiquinol:cytochrome c2 oxidoreductase in situ in Rhodopseudomonas palustris membranes

The presence of ubiquinol:cytochrome c2 oxidoreductase was shown in the membranes from a photosynthetic bacterium, Rhodopseudomonas palustris. Some properties of the enzyme in situ were investigated. The optimal pH of this enzyme activity was 7.0 in the intact membranes. The activity was inhibited by both antimycin and myxothiazol. Maximal activity (Vmax) was 3-4 mol cytochrome c (c2) reduced/mol cytochrome c1.s. Apparent activity of the enzyme with horse heart cytochrome c as the electron acceptor decreased as the concentration of salts in the reaction mixture increased, whereas when R. palustris cytochrome c2 was used as the electron acceptor, the activity increased as the concentration of salts increased. Moreover, the activity of the enzyme did not depend on the species or concentration of anions but on both the concentration and valency of the cations of the salts. These salt effects were thought to be due to the change of effective concentration of cytochrome molecules caused by cations near the membrane surface, which was net negatively charged. Apparent Km for ubiquinol-1 was about 80 microM irrespective of the species of cytochrome and the presence of salts.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes: evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 X g particulate fraction of the postnuclear supernatant obtained frm guinea-pig phagocytosing poymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O2- generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH. 2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1:1. 3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O2- generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation. 4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O2- formed during the oxidation of NADPH. 5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reactin at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH. 6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH. 7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible. 8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase. 9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O2; an enzymatic, apparently non-univalent, O2 reduction and a non-enzymatic chain reaction. 10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O2 is optimal at pH 7.0 with 1 mM NADPH.     

NADPH-cytochrome P-450 oxidoreductase. The role of cysteine 566 in catalysis and cofactor binding

Site-directed mutagenesis was employed to investigate the role of Cys566 in the catalytic mechanism of rat liver NADPH-cytochrome P-450 oxidoreductase. Rat NADPH-cytochrome P-450 oxidoreductase and mutants containing either alanine or serine at position 566 were expressed in Escherichia coli and purified to homogeneity. Substitution of alanine at position 566 had no effect on enzymatic activity with the acceptors cytochrome c and ferricyanide but did increase trans-hydrogenase activity with 3-acetylpyridine adenine dinucleotide phosphate by 79%. The Km for NADPH was increased 2.5-fold, and the NADP+ KI was increased 4.8-fold compared with that found for the wild-type enzyme. The conservative substitution, Ser566, produced a 50% decrease in cytochrome c reductase activity whereas activity with ferricyanide was decreased 57%, and 3-acetylpyridine adenine dinucleotide phosphate activity was unaffected. The NADPH Km was increased 4.6-fold, and the NADP+ KI increased 7.6-fold. The dependence of cytochrome c reductase activity on the KCl concentration was markedly altered by the Cys566 substitutions. Maximum activity for the wild-type enzyme was observed at approximately 0.18 M KCl whereas maximum activity for the mutant enzymes was observed between 0.04 and 0.09 M KCl. The pH dependence of cytochrome c reductase activity, cytochrome c Km, and flavin content were unaffected by these substitutions. These results demonstrate that Cys566 is not essential for activity of rat liver NADPH-cytochrome P-450 oxidoreductase although the cysteine side chain does affect the interaction of NADPH with the enzyme.