Redox Properties of Thermus thermophilus ba3: Different Electron-Proton Coupling in Oxygen Reductases?

A comprehensive study of the thermodynamic redox behavior of the hemes of the ba₃ enzyme from Thermus thermophilus, a B-type heme-copper oxygen reductase, is presented. This enzyme, in contrast to those having a single type of heme, allows the B- and A-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. The relative order of the midpoint reduction potentials of each center changed in the pH range from 6 to 8.4, and both hemes present a significant redox-Bohr effect. For instance, at pH 7, the midpoint reduction potentials of the hemes B and A₃ are 213 mV and 285 mV, respectively, whereas at pH 8.4, the order is reversed: 246 mV for heme B and 199 mV for heme A₃. The existence of redox anticooperativity was established by introducing a redox interaction parameter in a model of pairwise interacting redox centers.     

Thermodynamic redox behavior of the heme centers in A-type heme-copper oxygen reductases: comparison between the two subfamilies

The study of the thermodynamic redox behavior of the hemes from two members of the A family of heme-copper oxygen reductases, Paracoccus denitrificans aa₃ (A1 subfamily) and Rhodothermus marinus caa₃ (A2 subfamily) enzymes, is presented. At different pH values, midpoint reduction potentials and interaction potentials were obtained in the framework of a pairwise model for two interacting redox centers. In both enzymes, the hemes have different reduction potentials. For the A1-type enzyme, it was shown that heme a has a pH-dependent midpoint reduction potential, whereas that of heme a₃ is pH independent. For the A2-type enzyme the opposite was observed. The midpoint reduction potential of heme c from subunit II of the caa₃ enzyme was determined by fitting the data with a single-electron Nernst curve, and it was shown to be pH dependent. The results presented here for these A-type enzymes are compared with those previously obtained for representative members of the B and C families.     

Effects of metal ions in the CuB center on the redox properties of heme in heme-copper oxidases: spectroelectrochemical studies of an engineered heme-copper center in myoglobin

The electrochemical properties of an engineered heme-copper center in myoglobin have been investigated by UV-visible spectroelectrochemistry. In the cyanide-bridged, spin-coupled heme-copper center in an engineered myoglobin, the presence of Zn(II) in the Cu(B) center raises the heme reduction potential from -85 to 49 mV vs NHE. However, in the cyanide-free, spin-decoupled derivative of the same protein, the presence of Zn(II) in the Cu(B) center exerts little influence on the heme reduction potentials (77 and 80 mV vs NHE, respectively, in the absence and in the presence of Zn(II)). Similar trends have also been observed when copper ion is present in the Cu(B) center, although on a smaller scale, due to reduction of Cu(II) to Cu(I) prior to heme reduction. These results show that the presence of a metal ion in the designed Cu(B) center has a significant effect on the redox potential of heme Fe only when the two metal centers are coupled through a bridging ligand between the two metal centers, indicating that spin coupling plays an important role in redox potential regulation. In addition, the presence of a single positively charged Cu(I) center in the Cu(B) center resulted in a much lower increase (16 mV) in heme reduction potential than that of two positively charged Zn(II) (118 mV). Therefore, the heme reduction potential must be lowered after the first electron transfer to reduce heme Fe(3+)-Cu(B)(2+) to Fe(3+)-Cu(B)(+). To raise the heme reduction potential to make the second electron transfer (i.e., reduction of Fe(3+)-Cu(B)(+) to Fe(2+)-Cu(B)(+)) to be favorable, most likely a proton or decoupling of the heme-copper center is needed in the heme-copper site. These findings provide a strong argument for a thermodynamic driving force basis for redox-regulated proton transfer in heme-copper oxidases.     

A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases.

Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.     

Calculated coupling of transmembrane electron and proton transfer in dihemic quinol:fumarate reductase

The quinol:fumarate reductase of Wolinella succinogenes binds a low- and a high-potential heme b group in its transmembrane subunit C. Both hemes are part of the electron transport chain between the two catalytic sites of this redox enzyme. The oxidation-reduction midpoint potentials of the hemes are well established but their assignment in the structure has not yet been determined. By simulating redox titrations, using continuum electrostatics calculations, it was possible to achieve an unequivocal assignment of the low- and high-potential hemes to the distal and proximal positions in the structure, respectively. Prominent features governing the differences in midpoint potential between the two hemes are the higher loss of reaction field energy for the proximal heme and the stronger destabilization of the oxidized form of the proximal heme due to several buried Arg and Lys residues. According to the so-called "E-pathway hypothesis", quinol:fumarate reductase has previously been postulated to exhibit a novel coupling of transmembrane electron and proton transfer. Simulation of heme b reduction indicates that the protonation state of the conserved residue Glu C180, predicted to play a key role in this process, indeed depends on the redox state of the hemes. This result clearly supports the E-pathway hypothesis.     

Redox titration of all electron carriers of cytochrome c oxidase by Fourier transform infrared spectroscopy

Electrochemical redox titrations of cytochrome c oxidase from Paraccocus denitrificans were performed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The majority of the differential infrared absorption features may be divided into four groups, which correlate with the redox transitions of the four redox centers of the enzyme. Infrared spectroscopy has the advantage of allowing one to measure independent alterations in redox centers, which are not well separated, or even observed, by other spectroscopic techniques. We found 12 infrared bands that titrated with the highest observed midpoint redox potential (E(m) = 412 mV at pH 6.5) and which had a pH dependence of 52 mV per pH unit in the alkaline region. These bands were assigned to be linked to the Cu(B) center. We assigned bands to the Cu(A) center that showed a pH-independent E(m) of 250 mV. Two other groups of infrared differential bands reflected redox transitions of the two heme groups and showed a more complex behavior. Each of them included two parts, corresponding to high- and low-potential redox transitions. For the bands representing heme a, the ratio of high- to low-potential components was ca. 3:2; for heme a(3) this ratio was ca. 2:3. Taking into account the redox interactions between the hemes, these ratios yielded a difference in E(m) of 9 mV between the hemes (359 mV for heme a; 350 mV for heme a(3) at pH 8.0). The extent of the redox interaction between the hemes (-115 mV at pH 8.0) was found to be pH-dependent. The pH dependence of the E(m) values for the two hemes was the same and about two times smaller than the theoretical one, suggesting that an acid/base group binds a proton upon reduction of either heme. The applied approach allowed assignment of infrared bands in each of the four groups to vibrations of the hemes, ligands of the redox centers, amino acid residues, and/or protein backbone. For example, the well-known band shift at 1737/1746 cm(-)(1) corresponding to the protonated glutamic acid E278 correlated with oxidoreduction of heme a.     

Thermodynamic characterization of triheme cytochrome PpcA from Geobacter sulfurreducens: evidence for a role played in e-/H+ energy transduction

The facultative aerobic bacterium Geobacter sulfurreducens produces a small periplasmic c-type triheme cytochrome with 71 residues (PpcA) under anaerobic growth conditions, which is involved in the iron respiration. The thermodynamic properties of the PpcA redox centers and of a protonatable center were determined using NMR and visible spectroscopy techniques. The redox centers have negative and different reduction potentials (-162, -143, and -133 mV for heme I, III, and IV, respectively, for the fully reduced and protonated protein), which are modulated by redox interactions among the hemes (covering a range from 10 to 36 mV) and by redox-Bohr interactions (up to -62 mV) between the hemes and a protonatable center located in the proximity of heme IV. All the interactions between the four centers are dominated by electrostatic effects. The microscopic reduction potential of heme III is the one most affected by the oxidation of the other hemes, whereas heme IV is the most affected by the protonation state of the molecule. The thermodynamic properties of PpcA showed that pH strongly modulates the redox behavior of the individual heme groups. A preferred electron transfer pathway at physiologic pH is defined, showing that PpcA has the necessary thermodynamic properties to perform e-/H+ energy transduction, contributing to a H+ electrochemical potential gradient across the periplasmic membrane that drives ATP synthesis. PpcA is 46% identical in sequence to and shares a high degree of structural similarity with a periplasmic triheme cytochrome c7 isolated from Desulfuromonas acetoxidans, a bacterium closely related to the Geobacteracea family. However, the results obtained for PpcA are quite different from those published for D. acetoxidans c7, and the physiological consequences of these differences are discussed.     

EPR determination of interaction redox potentials in a multiheme cytochrome: cytochrome c3 from Desulfovibrio desulfuricans Norway

In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desulfuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40-50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.     

A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis

The macroscopic and microscopic redox potentials of the four hemes of the small tetraheme cytochrome c from Shewanella oneidensis were determined. The microscopic redox potentials show that the order of reduction is from hemes in the C-terminal domain (hemes 3 and 4) to the N-terminal domain (heme 1), demonstrating the polarization of the tetraheme chain during reduction. This makes heme 4 the most efficient electron delivery site. Furthermore, multi-step reduction of other redox centers through either heme 4 or heme 3 is shown to be possible. This has provided new insights into the two-electron reduction of the flavin in the homologous flavocytochrome c-fumarate reductase.     

Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior

Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center. Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1. Reproducible hysteresis is demonstrated in the redox titrations. In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV. However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2. A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated. In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1). The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes.     

Active site structure of the aa3 quinol oxidase of Acidianus ambivalens

The membrane bound aa(3)-type quinol:oxygen oxidoreductase from the hyperthermophilic archaeon, Acidianus ambivalens, which thrives at a pH of 2.5 and a temperature of 80 degrees C, has several unique structural and functional features as compared to the other members of the heme-copper oxygen reductase superfamily, but shares the common redox-coupled, proton-pumping function. To better understand the properties of the heme a(3)-Cu(B) catalytic site, a resonance Raman spectroscopic study of the enzyme under a variety of conditions and in the presence of various ligands was carried out. Assignments of several heme vibrational modes as well as iron-ligand stretching modes are made to serve as a basis for comparing the structure of the enzyme to that of other oxygen reductases. The CO-bound oxidase has conformations that are similar to those of other oxygen reductases. However, the addition of CO to the resting enzyme does not generate a mixed valence species as in the bovine aa(3) enzyme. The cyanide complex of the oxidized enzyme of A. ambivalens does not display the high stability of its bovine counterpart, and a redox titration demonstrates that there is an extensive heme-heme interaction reflected in the midpoint potentials of the cyanide adduct. The A. ambivalens oxygen reductase is very stable under acidic conditions, but it undergoes an earlier alkaline transition than the bovine enzyme. The A. ambivalens enzyme exhibits a redox-linked reversible conformational transition in the heme a(3)-Cu(B) center. The pH dependence and H/D exchange demonstrate that the conformational transition is associated with proton movements involving a group or groups with a pK(a) of approximately 3.8. The observed reversibility and involvement of protons in the redox-coupled conformational transition support the proton translocation model presented earlier. The implications of such conformational changes are discussed in relation to general redox-coupled proton pumping mechanisms in the heme-copper oxygen reductases.     

Electron transfer to the active site of the bacterial nitric oxide reductase is controlled by ligand binding to heme b₃

The active site of the bacterial nitric oxide reductase from Paracoccus denitrificans contains a dinuclear centre comprising heme b? and non heme iron (Fe(B)). These metal centres are shown to be at isopotential with midpoint reduction potentials of E(m) ≈ +80 mV. The midpoint reduction potentials of the other two metal centres in the enzyme, heme c and heme b, are greater than the dinuclear centre suggesting that they act as an electron receiving/storage module. Reduction of the low-spin heme b causes structural changes at the dinuclear centre which allow access to substrate molecules. In the presence of the substrate analogue, CO, the midpoint reduction potential of heme b? is raised to a region similar to that of heme c and heme b. This leads us to suggest that reduction of the electron transfer hemes leads to an opening of the active site which allows substrate to bind and in turn raises the reduction potential of the active site such that electrons are only delivered to the active site following substrate binding.     

Kinetic study of the reduction mechanism for Desulfovibrio gigas cytochrome c3.

The kinetic aspects of the reduction process in cytochrome c3 from Desulfovibrio gigas have been investigated over a wide range of pH values ranging between pH 5.8 and pH 9.8. The data have been analyzed in the framework of an I2H4 interaction network coupled to a proton-linked equilibrium between two tertiary structures (Cornish-Bowden, A. & Koshland, D.E. Jr (1970) J. Biol. Chem. 245, 6241-6250). The kinetic rate constants for the reduction of the four hemes for the two tertiary conformations have been characterized in the framework of the thermodynamic network obtained from the equilibrium analysis (Coletta, M., Catarino, T., LeGall, J.J. & Xavier, A.V. (1991) Eur. J. Biochem. 202, 1101-1106). The intrinsic reduction rate constants determined by reaction with sodium dithionite for two hemes (namely heme 4 and heme 1) are significantly faster than those for the other two heme residues. In view of the equilibrium redox properties, heme 4 (with the fastest reduction rate) may then work as the kinetic electron-capturing site for the electrons from sodium dithionite. The transfer to hemes 2 and 3 then occurs by virtue of their free-energy levels at equilibrium. At our experimental conditions, there is also transfer of electrons to hemes 2 and 3 from heme 1, which is reduced at a slower rate than heme 4, thus contributing to the biphasic kinetics observed for the overall process. The kinetic parameters obtained are discussed in terms of the mechanism proposed for the coupling between the electron and proton transfer, as induced by the heme/heme cooperativity network.     

Tuning heme redox potentials in the cytochrome C subunit of photosynthetic reaction centers

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.     

Stopped-flow and rapid-scan studies of the redox behavior of cytochrome aco from facultative alkalophilic Bacillus

Cytochrome aco purified from an alkalophilic bacterium grown at pH 10 contains hemes a, b, and c as prosthetic groups, and their redox behavior was examined by using stopped-flow and rapid-scan techniques. Under anaerobic conditions the reduction of both heme a and c moieties with dithionite proceeded exponentially but with different rates, usually the former being reduced about 4 times faster than the latter. The reduction of protoheme was much slower, and a time-difference spectrum for this species was of a high spin type with absorption peaks at 433, 557, and 609 nm. Only the protoheme combined with CO, fulfilling the criteria for cytochrome o. Potentiometric titrations determined a midpoint potential of c heme to be 95 mV at pH 7.0 and 25 degrees C and suggested the presence of two forms of a heme with midpoint potentials of 250 and 323 mV. Cytochrome aco utilizes ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) to reduce oxygen relatively rapidly without added cytochrome c (Qureshi, M. H., Yumoto, I., Fujiwara, T., Fukumori, Y., Yamanaka, T. (1990) J. Biochem. 107, 480-485). During the steady state, however, heme a stayed almost fully reduced in contrast to a partial reduction of heme c. Even after exhaustion of the dissolved oxygen the extent of reduction of heme c was 60-70% that attained by the dithionite reduction. When ascorbate plus TMPD-reduced cytochrome aco was exposed to oxygen the reduced heme c was oxidized rapidly whereas the oxidation of reduced a heme was negligibly slow. The full reduction of heme a during the steady state and its extremely slow oxidation rendered participation of heme a in the oxidase reaction less likely. A novel peak appearing transiently around 567 nm during the reaction was tentatively ascribed to an intermediate form of protoheme, or o heme, which was thus supposed to react directly with molecular oxygen. These results suggest strongly that the main electron transfer pathway would be c----o----oxygen. A possible role of a in regulating the electron flow through the main pathway and its functional relationship to a heme in the aa3-type cytochrome oxidase were discussed.     

EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli. Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism.

The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.     

The protonation state of a heme propionate controls electron transfer in cytochrome c oxidase

In cytochrome c oxidase (CcO), exergonic electron transfer reactions from cytochrome c to oxygen drive proton pumping across the membrane. Elucidation of the proton pumping mechanism requires identification of the molecular components involved in the proton transfer reactions and investigation of the coupling between internal electron and proton transfer reactions in CcO. While the proton-input trajectory in CcO is relatively well characterized, the components of the output pathway have not been identified in detail. In this study, we have investigated the pH dependence of electron transfer reactions that are linked to proton translocation in a structural variant of CcO in which Arg481, which interacts with the heme D-ring propionates in a proposed proton output pathway, was replaced with Lys (RK481 CcO). The results show that in RK481 CcO the midpoint potentials of hemes a and a(3) were lowered by approximately 40 and approximately 15 mV, respectively, which stabilizes the reduced state of Cu(A) during reaction of the reduced CcO with O(2). In addition, while the pH dependence of the F --> O rate in wild-type CcO is determined by the protonation state of two protonatable groups with pK(a) values of 6.3 and 9.4, only the high-pK(a) group influences this rate in RK481 CcO. The results indicate that the protonation state of the Arg481 heme a(3) D-ring propionate cluster having a pK(a) of approximately 6.3 modulates the rate of internal electron transfer and may act as an acceptor of pumped protons.     

Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.

Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.