MacA, a diheme c-type cytochrome involved in Fe(III) reduction by Geobacter sulfurreducens

A 36-kDa diheme c-type cytochrome abundant in Fe(III)-respiring Geobacter sulfurreducens, designated MacA, was more highly expressed during growth with Fe(III) as the electron acceptor than with fumarate. Although MacA has homology to proteins with in vitro peroxidase activity, deletion of macA had no impact on response to oxidative stress. However, the capacity for Fe(III) reduction was greatly diminished, indicating that MacA, which is predicted to be localized in the periplasm, is a key intermediate in electron transfer to Fe(III).     

Geobacter sulfurreducens cytochrome c peroxidases: electrochemical classification of catalytic mechanisms

Bacterial cytochrome c peroxidase (CcP) enzymes are diheme redox proteins that reduce hydrogen peroxide to water. They are canonically characterized by a peroxidatic (called L, for "low reduction potential") active site heme and a secondary heme (H, for "high reduction potential") associated with electron transfer, and an enzymatic activity that exists only when the H-heme is prereduced to the Fe(II) oxidation state. The prereduction step results in a conformational change at the active site itself, where a histidine-bearing loop will adopt an "open" conformation allowing hydrogen peroxide to bind to the Fe(III) of the L-heme. Notably, the enzyme from Nitrosomonas europaea does not require prereduction. Previously, we have shown that protein film voltammetry (PFV) is a highly useful tool for distinguishing the electrocatalytic mechanisms of the Nitromonas type of enzyme from other CcPs. Here, we apply PFV to the recently described enzyme from Geobacter sulfurreducens and the Geobacter S134P/V135K double mutant, which have been shown to be similar to members of the canonical subclass of peroxidases and the Nitrosomonas subclass of enzymes, respectively. Here we find that the wild-type Geobacter CcP is indeed similar electrochemically to the bacterial CcPs that require reductive activation, yet the S134P/V135K mutant shows two phases of electrocatalysis: one that is low in potential, like that of the wild-type enzyme, and a second, higher-potential phase that has a potential dependent upon substrate binding and pH yet is at a potential that is very similar to that of the H-heme. These findings are interpreted in terms of a model in which rate-limiting intraprotein electron transfer governs the catalytic performance of the S134P/V135K enzyme.     

The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation

We report the characterization of the diheme cytochrome c peroxidase (CcP) from Shewanella oneidensis (So) using UV-visible absorbance, electron paramagnetic resonance spectroscopy, and Michaelis-Menten kinetics. While sequence alignment with other bacterial diheme cytochrome c peroxidases suggests that So CcP may be active in the as-isolated state, we find that So CcP requires reductive activation for full activity, similar to the case for the canonical Pseudomonas type of bacterial CcP enzyme. Peroxide turnover initiated with oxidized So CcP shows a distinct lag phase, which we interpret as reductive activation in situ. A simple kinetic model is sufficient to recapitulate the lag-phase behavior of the progress curves and separate the contributions of reductive activation and peroxide turnover. The rates of catalysis and activation differ between MBP fusion and tag-free So CcP and also depend on the identity of the electron donor. Combined with Michaelis-Menten analysis, these data suggest that So CcP can accommodate electron donor binding in several possible orientations and that the presence of the MBP tag affects the availability of certain binding sites. To further investigate the structural basis of reductive activation in So CcP, we introduced mutations into two different regions of the protein that have been suggested to be important for reductive activation in homologous bacterial CcPs. Mutations in a flexible loop region neighboring the low-potential heme significantly increased the activation rate, confirming the importance of flexible loop regions of the protein in converting the inactive, as-isolated enzyme into the activated form.     

CcpA from Geobacter sulfurreducens Is a Basic Di-Heme Cytochrome c Peroxidase

Bacterial di-heme cytochrome c peroxidases (CcpAs) protect the cell from reactive oxygen species by reducing hydrogen peroxide to water. The enzymes are c-type cytochromes, with both heme groups covalently attached to the protein chain via a characteristic binding motif. The genome of the dissimilatory metal-reducing bacterium Geobacter sulfurreducens revealed the presence of a ccpA gene and we isolated the gene product after recombinant expression in Escherichia coli. CcpA from G. sulfurreducens exhibited in vitro peroxidase activity with ABTS²⁻ [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] as an electron donor, and the three-dimensional structure of the dimeric enzyme has been determined to high resolution. For activation, CcpA commonly requires reduction, with the exception of the Nitrosomonas europaea enzyme that retains its activity in the oxidized state. A G94K/K97Q/R100I triple point mutant was created to mimic the critical loop region of N. europaea CcpA, but its crystal structure revealed that the inactive, bis-histidinyl-coordinated form of the active-site heme group was retained. Subsequent mutational studies thus addressed an adjacent loop region, where a change in secondary structure accompanies the reductive activation of the enzyme. While an A124K/K128A double mutant did not show significant changes, the CcpA variants S134P/V135K and S134P led to a distortion of the loop region, accompanied by an opening of the active-site loop, leaving the enzyme in a constitutively active state.     

Functional importance of tyrosine 294 and the catalytic selectivity for the bis-Fe(IV) state of MauG revealed by replacement of this axial heme ligand with histidine

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high-valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His, and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in interprotein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation affects not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 ? separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high-valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)═O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)═O heme but via long-range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high-valent heme iron.     

Hydrophobic modulation of heme properties in heme protein maquettes

We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each alpha-helix, alpha-SH, in [I(6)F(13)H(24)](2) is Ac-CGGGEI(6)WKL.H(10)EEF(13)LKK.FEELLKL.H(24)EERLKK.L-CONH(2). In our nomenclature, [I(6)F(13)H(24)] represents the disulfide-bridged di-alpha-helical homodimer of this sequence, i.e., (alpha-SS-alpha), and [I(6)F(13)H(24)](2) represents the dimeric four helix bundle composed of two di-alpha-helical subunits, i.e., (alpha-SS-alpha)(2). We replaced the histidines at positions H24 in [I(6)F(13)H(24)](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I(6)F(13)I(24)](2), [I(6)F(13)A(24)](2), and [I(6)F(13)F(24)](2), are distinguished from the tetraheme binding parent peptide, [I(6)F(13)H(24)](2), by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I(6)F(13)F(24)](2) maquette was related to the first generation diheme [H10A24](2) prototype, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H)2(O) >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K(d1) value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K(d2) in the range of 35-800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K(d) values. The equilibrium reduction midpoint potential (E(m7.5)) determined in the monoheme state ranges from -156 to -210 mV vs SHE and in the diheme state ranges from -144 to -288 mV. An observed heme-heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global topology of the di-alpha-helical monomers. The heme affinity and electrochemistry of the three H24 variants studied identify the tight binding sites (K(d1) and K(d2) values <200 nM) having the lower reduction midpoint potentials (E(m7.5) values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2). The results of this study illustrate that conservative hydrophobic amino acid changes near the heme binding site can modulate the E(m) by up to +/-50 mV and the K(d) by an order of magnitude. Furthermore, the effects of multiple single amino acid changes on E(m) and K(d) do not appear to be additive.     

The distal heme center in Bacillus subtilis succinate:quinone reductase is crucial for electron transfer to menaquinone

Succinate:quinone reductases are membrane-bound enzymes that catalyze electron transfer from succinate to quinone. Some enzymes in vivo reduce ubiquinone (exergonic reaction) whereas others reduce menaquinone (endergonic reaction). The succinate:menaquinone reductases all contain two heme groups in the membrane anchor of the enzyme: a proximal heme (heme b(P)) located close to the negative side of the membrane and a distal heme (heme b(D)) located close to the positive side of the membrane. Heme b(D) is a distinctive feature of the succinate:menaquinone reductases, but the role of this heme in electron transfer to quinone has not previously been analyzed. His28 and His113 are the axial ligands to heme b(D) in Bacillus subtilis succinate:menaquinone reductase. We have individually replaced these His residues with Leu and Met, respectively, resulting in assembled membrane-bound enzymes. The H28L mutant enzyme lacks succinate:quinone reductase activity probably due to a defective quinone binding site. The H113M mutant enzyme contains heme b(D) with raised midpoint potential and is impaired in electron transfer to menaquinone. Our combined experimental data show that the heme b(D) center, into which we include a quinone binding site, is crucial for succinate:menaquinone reductase activity. The results support a model in which menaquinone is reduced on the positive side of the membrane and the transmembrane electrochemical potential provides driving force for electron transfer from succinate via heme b(P) and heme b(D) to menaquinone.     

Cytochrome c-dependent methacrylate reductase from Geobacter sulfurreducens AM-1.

Geobacter sulfurreducens AM-1 can use methacrylate as a terminal electron acceptor for anaerobic respiration. In this paper, we report on the purification and properties of the periplasmic methacrylate reductase, and show that the enzyme is dependent on the presence of a periplasmic cytochrome c (apparent K(m) = 0.12 microM). The methacrylate reductase was found to be composed of only one polypeptide with an apparent molecular mass of 50 kDa and to contain, bound tightly but not covalently, 1 mol of FAD per mol. The N-terminal amino acid sequence showed sequence similarity to a periplasmic fumarate reductase from Shewanella putrefaciens. However, methacrylate reductase did not catalyze the reduction of fumarate. The periplasmic cytochrome c, which was also purified, had an apparent molecular mass of 30 kDa and contained approximately 4 mol of heme.mol(-1). Cells of G. sulfurreducens AM-1 grown on acetate and methacrylate as an energy source were found to contain all the enzymes required for the oxidation of acetate to CO(2) via the citric acid cycle.     

Structural and biochemical characterization of DHC2, a novel diheme cytochrome c from Geobacter sulfurreducens

Multiheme cytochromes c constitute a widespread class of proteins with essential functions in electron transfer and enzymatic catalysis. Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs. Understanding the significance of these motifs is crucial for the elucidation of the highly optimized properties of multiheme cytochromes c, but their spectroscopic investigation is often hindered by the large number and efficient coupling of the individual centers and the limited availability of recombinant protein material. We have identified a diheme cytochrome c, DHC2, from the metal-reducing soil bacterium Geobacter sulfurreducens and determined its crystal structure by the method of multiple-wavelength anomalous dispersion (MAD). The two heme groups of DHC2 pack into one of the typical heme interaction motifs observed in larger multiheme cytochromes, but because of the absence of further, interfering cofactors, the properties of this heme packing motif can be conveniently studied in detail. Spectroscopic properties (UV-vis and EPR) of the protein are typical for cytochromes containing low-spin Fe(III) centers with bis-histidinyl coordination. Midpoint potentials for the two heme groups have been determined to be -135 and -289 mV by potentiometric redox titrations. DHC2 has been produced by recombinant expression in Escherichia coli using the accessory plasmid pEC86 and is therefore accessible for systematic mutational studies in further investigating the properties of heme packing interactions in cytochromes c.     

The conserved Trp-Cys hydrogen bond dampens the "push effect" of the heme cysteinate proximal ligand during the first catalytic cycle of nitric oxide synthase

Residues surrounding and interacting with the heme proximal ligand are important for efficient catalysis by heme proteins. The nitric oxide synthases (NOSs) are thiolate-coordinated enzymes that catalyze the hydroxylation of l-Arg in the first of the two catalytic cycles needed to synthesize nitric oxide. In NOSs, the indole NH group of a conserved tryptophan [W56 of the bacterial NOS-like protein from Staphylococcus aureus (saNOS)] forms a hydrogen bond with the heme proximal cysteinate ligand. The purpose of this study was to determine the impact of increasing (W56F and W56Y variants) or decreasing (W56H variant) the electron density of the proximal cysteinate ligand on molecular oxygen (O(2)) activation using saNOS as a model. We show that the removal of the indole NH···S(-) bond for W56F and W56Y caused an increase in the electron density of the cysteinate. This was probed by the decrease of the midpoint reduction potential (E(1/2)) along with weakened σ-bonding and strengthened π-backbonding with distal ligands (CO and O(2)). On the other hand, the W56H variant showed stronger Fe-OO and Fe-CO bonds (strengthened σ-bonding) along with an elevated E(1/2), which is consistent with the formation of a strong NH···S(-) hydrogen bond from H56. We also show here that changing the electron density of the proximal thiolate controls its "push effect"; whereas the rates of both O(2) activation and autoxidation of the Fe(II)O(2) complex increase with the stronger push effect created by removing the indole NH···S(-) hydrogen bond (W56F and W56Y variants), the W56H variant showed an increased stability of the complex against autoxidation and a slower rate of O(2) activation. These results are discussed with regard to the roles played by the conserved tryptophan-cysteinate interaction in the first catalytic cycle of NOS.     

Structure-function studies on nitric oxide synthases

Nitric oxide synthase (NOS) catalyzes the oxidation of one l-arginine guanidinium N atom to nitric oxide (NO). NOS consists of a heme domain linked to a flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD) reductase that shuttles electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to the heme. This review summarizes various aspects of NOS structure and function derived from crystal structures coupled with a wealth of biochemical and biophysical data. This includes the binding of diatomic ligands, especially the product, NO, whose binding to the heme iron blocks enzyme activity. An unusual feature of NOS catalysis is the strict requirement for the essential cofactor, tetrahydrobiopterin (H4B). It now is generally agreed that H4B serves as an electron donor to the heme-oxy complex. The reason NOS may have recruited H4B as an electron transfer cofactor is to provide rapid coupled proton/electron transfer required for O2 activation. NOS is a highly regulated enzyme which is controlled by calmodulin (CaM) at the level of electron transfer within the FMN/FAD reductase and between the reductase and heme domains. Recent crystal structures provide a basis for developing models on the structural underpinnings of NOS regulation. In addition to the complex and fascinating functional and regulatory features of NOS, NOS is an important therapeutic target. Crystal structures have revealed the structural basis of isoform-selective inhibition by a group of dipeptide inhibitors which opens the way for structure-based inhibitor design.     

Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd 1 nitrite reductase: regulation of the NO-bound end product

The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome C553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c(552red)-->NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1+/-0.1x10(5) M(-1) s(-1) (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s(-1) could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4+/-0.7 s(-1), corresponding to a k2 value of 2.2+/-1.1x10(4) M(-1) s(-1) for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation.     

Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa. The role of a heme-linked protein loop: a mutagenesis study

Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67-79) in the activation of di-heme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c(551). Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67-79 in the activation of di-heme peroxidases and suggest a "charge hopping" mechanism may be operative in the process of intra-molecular electron transfer.     

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.     

Histidine 61: an important heme ligand in the soluble fumarate reductase from Shewanella frigidimarina

An examination of the X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112] shows the presence of four, bis-His-ligated, c-type hemes and one flavin adenine dinucleotide, FAD. The heme groups provide a "molecular wire" for the delivery of electrons to the FAD. Heme IV is closest to the FAD (7.4 A from heme methyl to FAD C7), and His61, a ligand to heme IV, is also close (8.4 A to FAD C7). Electron delivery to the FAD from the heme groups must proceed via heme IV, as hemes I-III are too far from the FAD for feasible electron transfer. To examine the importance of heme IV and its ligation for enzyme function, we have substituted His61 with both methionine and alanine. Here we describe the crystallographic, kinetic, and electrochemical characterization of the H61M and H61A mutant forms of the Shewanella fumarate reductase. The crystal structures of these mutant forms of the enzyme have been determined to 2.1 and 2.2 A resolution, respectively. Substitution of His61 with alanine results in heme IV having only one protein ligand (His86), the sixth coordination position being occupied by an acetate ion derived from the crystal cryoprotectant solution. In the structure of the H61M enzyme, Met61 is found not to ligate the heme iron, a role that is taken by a water molecule. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the H61M-Fcc(3) and H61A-Fcc(3) mutant enzymes are catalytically active but exhibit marked decreases in the value of k(cat) for fumarate reduction with respect to that of the wild type (5- and 10-fold lower, respectively). There is also a significant shift in the pK(a) values for the mutant enzymes, from 7.5 for the wild type to 8.26 for H61M and 9.29 for H61A. The fumarate reductase activity of both mutant enzymes can be recovered to approximately 80% of that seen for the wild type by the addition of exogenous imidazole. In the case of H61A, recovery of activity is also accompanied by a shift of the pK(a) from 9.29 to 7.46 (close, and within experimental error, to that for the wild type). Pre-steady-state kinetic measurements show clearly that rate constants for the fumarate dependent reoxidation of the heme groups are adversely affected by the mutations. The solvent isotope effect for fumarate reduction in the wild-type enzyme has a value of 8.0, indicating that proton delivery is substantially rate limiting. This value falls to 5.6 and 2.2 for the H61M and H61A mutants, respectively, indicating that electron transfer, rather than proton transfer, is becoming more rate-limiting in the mutant enzymes.     

P450BM-3: reduction by NADPH and sodium dithionite.

Microsomal P450s catalyze the monooxygenation of a large variety of hydrophobic compounds, including drugs, steroids, carcinogens, and fatty acids. The interaction of microsomal P450s with their electron transfer partner, NADPH-P450 reductase, during the transfer of electrons from NADPH to P450, for oxygen activation, may be important in regulating this enzyme system. Highly purified Bacillus megaterium P450BM-3 is catalytically self-sufficient and contains both the reductase and P450 domains on a single polypeptide chain of approximately 120,000 Da. The two domains of P450BM-3 appear to be analogous in their function and homologous in their sequence to the microsomal P450 system components. FAD, FMN, and heme residues are present in equimolar amounts in purified P450BM-3 and, therefore, this protein could potentially accept five electron equivalents per mole of enzyme during a reductive titration. The titration of P450BM-3 with sodium dithionite under a carbon monoxide atmosphere was complete with the addition of the expected five electron equivalents. The intermediate spectra indicate that the heme iron is reduced first, followed by the flavin residues. Titration of the protein with the physiological reductant, NADPH, also required approximately five electron equivalents when the reaction was performed under an atmosphere of carbon monoxide. Under an atmosphere of argon and in the absence of carbon monoxide, one of the flavin groups was reduced prior to the reduction of the heme group. The titration behavior of P450BM-3 with NADPH was surprising because no spectral changes characteristic of flavin semiquinone intermediates were observed. The results of the titration with NADPH can only be explained if (a) there was "rapid" intermolecular electron transfer between P450BM-3 molecules, (b) there is no kinetic barrier to the reduction of P450 by the one-electron-reduced form of the reductase, and (c) the "air-stable semiquinone" form of the reductase does not accumulate in this complex multidomain enzyme.