Flavin-sensitized photoreduction of thymidine glycol

Photochemical reactivity of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) toward thymidine glycol (dTg) has been investigated. Fluorescence intensity of FAD was enhanced as increasing the concentration of dTg, suggesting that adenosine moiety of FAD interacts with dTg. However, photoreduction of dTg using reduced form of FAD gave repaired thymidine in almost the same yield as when reduced FMN was used alternatively, and thus such interaction seems to have no effect on the reduction. Oligodeoxynucleotides containing dTg were also photochemically repaired by reduced form of flavins in different yields depending on the sequence, which could be related to electron affinity of the nucleobases in DNA.     

Tryptophan 697 modulates hydride and interflavin electron transfer in human methionine synthase reductase

Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins. We created four MSR mutants (W697S, W697H, S698Δ, and S698A) and studied their associated kinetic behavior. Multiwavelength stopped-flow analysis reveals that NADPH reduction of the C-terminal Ser698 mutants occurs in three resolvable kinetic steps encompassing transfer of a hydride ion to FAD, semiquinone formation (indicating FAD to FMN electron transfer), and slow flavin reduction by a second molecule of NADPH. Corresponding experiments with the W697 mutants show a two-step flavin reduction without an observable semiquinone intermediate, indicating that W697 supports FAD to FMN electron transfer. Accelerated rates of FAD reduction, steady-state cytochrome c(3+) turnover, and uncoupled NADPH oxidation in the S698Δ and W697H mutants may be attributed to a decrease in the energy barrier for displacement of W697 by NADPH. Binding of NADP(+), but not 2',5'-ADP, is tighter for all mutants than for native MSR. The combined studies demonstrate that while W697 attenuates hydride transfer, it ensures coenzyme selectivity and accelerates FAD to FMN electron transfer. Moreover, analysis of analogous cytochrome P450 reductase (CPR) variants points to key differences in the driving force for flavin reduction and suggests that the conserved FAD stacking tryptophan residue in CPR also promotes interflavin electron transfer.     

The Fragments of the Photosynthetic Electron Transfer Chain in Model Systems

In this paper the recent research from our laboratory is reviewed. Short fragments of the photochemical electron transfer chain of photosynthesis were reproduced in aqueous detergent solutions or in organic solvents. The function of photosystem I is reproduced in a ternary system of chlorophylls, electron donors (dienols, sulfhydryl compounds, hydrazine, etc.), and electron acceptors (viologens, nicotinamide-adenine dinucleotide [NAD], flavines, etc.). Chlorophyll-photosensitized reduction of viologens in some cases is activated by oxygen at the expense of active reductants formed during the photosensitized oxidation of an initial electron donor (thiourea). Chlorophyll-photosensitized oxidoreduction of cytochromes is activated by flavines, viologens, vitamin K derivatives, and some other redox systems (cofactors of cyclic photophosphorylation). The primary mechanism of the reactions studied depends on the reversible chlorophyll photooxidoreduction. In binary systems, chlorophyll (monomeric or aggregated) and electron donor or electron acceptor, reversible photoreduction or photooxidation is observed. Irreversible bacteriochlorophyll oxidation leads to the formation of chlorophyll and protochlorophyll analogues; irreversible protochlorophyll photoreduction results in chlorophyll-like pigment appearance. The photodisaggregation of chlorophyll was observed. The models of photosystem II studied were the photochemical oxygen evolution in aqueous solutions of electron acceptors (ferric compounds, quinone), photosensitized in the near UV part of the spectrum by inorganic semiconductors (tungsten, titanium, and zinc oxides). All reactions described are based on electron (hydrogen) transfer photosensitized by pigment system.     

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.     

Role of reductase domain cluster 1 acidic residues in neuronal nitric-oxide synthase. Characterization of the FMN-FREE enzyme.

The nNOS reductase domain is homologous to cytochrome P450 reductase, which contains two conserved clusters of acidic residues in its FMN module that play varied roles in its electron transfer reactions. To study the role of nNOS reductase domain cluster 1 acidic residues, we mutated two conserved acidic (Asp(918) and Glu(919)) and one conserved aromatic residue (Phe(892)), and investigated the effect of each mutation on flavin binding, conformational change, electron transfer reactions, calmodulin regulation, and catalytic activities. Each mutation destabilized FMN binding without significantly affecting other aspects including substrate, cofactor or calmodulin binding, or catalytic activities upon FMN reconstitution, indicating the mutational effect was restricted to the FMN module. Characterization of the FMN-depleted mutants showed that bound FMN was essential for reduction of the nNOS heme or cytochrome c, but not for ferricyanide or dichlorophenolindolphenol, and established that the electron transfer path in nNOS is NADPH to FAD to FMN to heme. Steady-state and stopped-flow kinetic analysis revealed a novel role for bound FMN in suppressing FAD reduction by NADPH. The suppression could be relieved either by FMN removal or calmodulin binding. Calmodulin binding induced a conformational change that was restricted to the FMN module. This increased the rate of FMN reduction and triggered electron transfer to the heme. We propose that the FMN module of nNOS is the key positive or negative regulator of electron transfer at all points in nNOS. This distinguishes nNOS from other related flavoproteins, and helps explain the mechanism of calmodulin regulation.     

A novel two-protein component flavoprotein hydroxylase.

p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) was purified from Acinetobacter baumannii and shown to be a two-protein component enzyme. The small component (C1) is the reductase enzyme with a subunit molecular mass of 32 kDa. C1 alone catalyses HPA-stimulated NADH oxidation without hydroxylation of HPA. C1 is a flavoprotein with FMN as a native cofactor but can also bind to FAD. The large component (C2) is the hydroxylase component that hydroxylates HPA in the presence of C1. C2 is a tetrameric enzyme with a subunit molecular mass of 50 kDa and apparently contains no redox centre. FMN, FAD, or riboflavin could be used as coenzymes for hydroxylase activity with FMN showing the highest activity. Our data demonstrated that C2 alone was capable of utilizing reduced FMN to form the product 3,4-dihydroxyphenylacetate. Mixing reduced flavin with C2 also resulted in the formation of a flavin intermediate that resembled a C(4a)-substituted flavin species indicating that the reaction mechanism of the enzyme proceeded via C(4a)-substituted flavin intermediates. Based on the available evidence, we conclude that the reaction mechanism of HPAH from A. baumannii is similar to that of bacterial luciferase. The enzyme uses a luciferase-like mechanism and reduced flavin (FMNH2, FADH2, or reduced riboflavin) to catalyse the hydroxylation of aromatic compounds, which are usually catalysed by FAD-associated aromatic hydroxylases.     

Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase

Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed‐function oxidase system that participates in the metabolism of endo‐ and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD‐to‐FMN, but not FMN‐to‐P450, electron transfer. Here, we report the 2.5 Å resolution crystal structure of a functionally competent yeast–human chimeric CPR in an open conformation, compatible with FMN‐to‐P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 Å.     

Determination of the redox properties of human NADPH-cytochrome P450 reductase

Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) [oxidized/semiquinone] = -286 +/- 6 mV, E(2) [semiquinone/reduced] = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) [FMN] = -66 +/- 8 mV, E(2) [FMN] = -269 +/- 10 mV; E(1) [FAD] = -283 +/- 5 mV, E(2) [FAD] = -382 +/- 8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples. Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase. However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes.     

Dihydropyrimidine dehydrogenase: a flavoprotein with four iron-sulfur clusters

Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme in the pathway for degradation of pyrimidines, responsible for the reduction of the 5,6-double bond to give the dihydropyrimidine using NADPH as the reductant. The enzyme is a dimer of 220 kDa, and each monomer contains one FAD, one FMN, and four FeS clusters. The FAD is situated at one end of the protein, the FMN is at the other, and four FeS clusters form a conduit for electron transfer between the two sites comprised of two FeS clusters from each monomer. The enzyme has a two-site ping-pong mechanism with NADPH reducing FAD and reduced FMN responsible for reducing the pyrimidine. Solvent deuterium kinetic isotope effects indicate a rate-limiting reduction of FAD accompanied by pH-dependent structural rearrangement for proper orientation of the nicotinamide ring. Transfer of electrons from site 1 to site 2 is downhill with FMN rapidly reduced by FADH(2) via the FeS conduit. The reduction of the pyrimidine at site 2 proceeds using general acid catalysis with protonation at N5 of FMN carried out by K574 as FMN is reduced and protonation at C5 of the pyrimidine by C671 as it is reduced. Kinetic isotope effects indicate a stepwise reaction for reduction of the pyrimidine with hydride transfer at C6 preceding proton transfer at C5, with a late transition state for the proton transfer step.     

Properties of the stable aerobic and anaerobic half-reduced states of NADPH-cytochrome c reductase.

The microsomal flavoprotein, NADPH-cytochrome c reductase, has been reexamined to determine: (1) the nature of the flavine bound to the enzyme and (2) the oxidation-reduction state of the "half-reduced" form of the flavoprotein. Iyanagi and Mason (Iyanagi, T., and Mason, H.S. (1973), Biochemistry 12, 2297) have recently proposed that NADPH-cytochrome c reductase contains both FAD and FMN as prosthetic groups in lieu of FAD as the sole constituent, as suggested by all previous studies of this enzyme. The data presented herein, utilizing the recently published fluorometric procedure of Faeder and Siegel (Faeder, E. J., and Siegle, L. M. (1973), Anal. Biochem. 53, 332) for the determination of FAD and FMN in mixtures, confirm the conclusions of Iyanagi and Mason for both rat and pig liver reductase preparations. Data for other flavoproteins are also presented. Iyanagi and Mason have also concluded that the air-stable "semiquinone" is a form of NADPH-cytochrome c reductase reduced by one electron per two falvines (F-FH). The present studies, however, do not agree with this conclusion, but instead support our previous results which indicate that both the aerobic and anaerobic half-reduced states of this flavoprotein exist in the two-electron reduced form (FH-FH). Removal of NADP+ does not affect the spectrum of the air-stable half-reduced form of the flavoprotein, nor does it affect the back titration of this intermediate by potassium ferricyanide. The possible implications of these observations on the catalytic cycle of the flavines of NADPH-cytochrome c reductase are discussed.     

Benzylidenemalononitrile derivatives as substrates and inhibitors of a new NAD(P)H dehydrogenase of erythrocytes. Purification and crystallisation of two forms of the enzyme.

Using the powerful lachrymator (2-chlorobenzylidene)malononitrile as electron acceptor, two types of NAD(P)H dehydrogenases have been isolated from human blood. Crystallisation of the homogenous enzymes was performed in 50% polyethylene glycol solution. The enzymes (average molecular weight 18 000) are composed of only one polypeptide chain and have a very similar amino acid composition. B-side stereospecificity was determined with respect to the cofactor by gas chromatography-mass spectrometry for the reductase. Besides (2-chlorobenzylidene)malononitrile, 2,6-dichloroindophenol, methylene blue, 4-benzoquinone, FMN and FAD are also reduced using NADH or NADPH as hydrogen donor with the rates decreasing in the given order. Reduction of methemoglobin is observed only upon addition of methylene blue, FMN or FAD as carriers. (2-Chlorobenzylidene)malononitrile reduction is inhibited by most of the compounds known to be decouplers of oxidative phosphorylation.     

NADPH-cytochrome P-450 reductase. Physical properties and redox behavior in the absence of the FAD moiety

NADPH-cytochrome P-450 reductase contains one molecule each of FMN and FAD. The FAD moiety has been selectively removed, producing the FMN reductase. The FMN reductase is stable and enzymatic activity is reconstituted with either FAD or FMN. FMN remains tightly bound, but can both dissociate from the FMN site and bind to the vacant FAD site. The amount of FMN bound in the FAD site is minimal under specific experimental conditions. There are at least two conformational subpopulations of the FMN reductase; NADP dissociates readily from one but extremely slowly from the other. Rapid dissociation of NADP is regained upon reconstitution with FAD. The one-electron redox state of the FMN reductase is thermodynamically stabilized, though to a lesser degree than in the holoreductase. When two-electron reduced FMN reductase is exposed to oxygen, a stable species with an absorbance peak at 580 nm forms rapidly and quantitatively. This species has been identified by electron paramagnetic resonance spectroscopy as the neutral radical of FMN and is indistinguishable from the air-stable radical of the holoreductase. The redox behavior of the FMN reductase is in agreement with properties proposed previously for the FMN site.     

Role of the conserved phenylalanine 181 of NADPH-cytochrome P450 oxidoreductase in FMN binding and catalytic activity.

NADPH-cytochrome P450 oxidoreductase (P450 reductase, EC 1.6.2.4) is an essential component of the P450 monooxygenase complex and binds FMN, FAD, and NADPH cofactors. Residues Tyr140 and Tyr178 are known to be involved in FMN binding. A third aromatic side chain, Phe181, is also located in the proximity of the FMN ring and is highly conserved in FMN-binding proteins, suggesting an important functional role. This role has been investigated by site-directed mutagenesis. Substitution of Phe181 with leucine or glutamine decreased the cytochrome c reductase activity of the enzyme by approximately 50%. Ferricyanide reductase activity was unaffected, indicating that the FAD domain was unperturbed. The mutant FMN domains were expressed in Escherichia coli, and the redox potentials and binding energies of their complexes with FMN were determined. The affinity for FMN was decreased approximately 50-fold in the Leu181 and Gln181 mutants. Comparison of the binding energies of the wild-type and mutant enzymes in the three redox states of FMN suggests that Phe181 stabilizes the FMN-apoprotein complex. The amide 1H and 15N resonances of the Phe181Leu FMN domain were assigned; comparison of their chemical shifts with those of the wild-type domain indicated that the effect of the substitution on FMN affinity results from perturbation of two loops which form part of the FMN binding site. The results indicate that Phe181 cooperates with Tyr140 and Tyr178 to play a major role in the binding and stability of FMN.     

The thermochemical characterization of sodium dithionite, flavin mononucleotide, flavin-adenine dinucleotide and methyl and benzyl viologens as low-potential reductants for biological systems.

The heat of reaction (deltaH) of Fe(CN)63-, Methyl Viologen, FMN and FAD with S2O42- in aqueous buffer solutions was measured calorimetrically. In addition deltaH values for reduction of Fe(CN)63-, FMN and FAD by reduced Methyl Viologen were determined. The resulting calorimetric data and corresponding E0 values were combined to yield thermodynamic data for these simple reducing agents in a form useful for applications to biological reactions. Thermodynamic data for the reduction of spinach ferredoxin are also presented.     

Identification and characterization of 2‐naphthoyl‐coenzyme A reductase,the prototype of a novel class of dearomatizing reductases

The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl‐coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl‐CoA by ATP‐dependent or ‐independent benzoyl‐CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2‐naphthoyl‐CoA reductase was purified from extracts of the naphthalene‐degrading, sulphidogenic enrichment culture N47. The oxygen‐tolerant enzyme dearomatized the non‐activated ring of 2‐naphthoyl‐CoA by a four‐electron reduction to 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin‐containing ‘old yellow enzyme’ family. NCR contained FAD, FMN, and an iron‐sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2‐naphthoyl‐CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl‐CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl‐CoA reductases.     

Redox properties of the isolated flavin mononucleotide- and flavin adenine dinucleotide-binding domains of neuronal nitric oxide synthase

Electron transfer through neuronal nitric oxide synthase (nNOS) is regulated by the reversible binding of calmodulin (CaM) to the reductase domain of the enzyme, the conformation of which has been shown to be dependent on the presence of substrate, NADPH. Here we report the preparation of the isolated flavin mononucleotide (FMN)-binding domain of nNOS with bound CaM and the electrochemical analysis of this and the isolated flavin adenine dinucleotide (FAD)-binding domain in the presence and absence of NADP(+) and ADP (an inhibitor). The FMN-binding domain was found to be stable only in the presence of bound CaM/Ca(2+), removal of which resulted in precipitation of the protein. The FMN formed a kinetically stabilized blue semiquinone with an oxidized/semiquinone reduction potential of -179 mV. This is 80 mV more negative than the potential of the FMN in the isolated reductase domain, that is, in the presence of the FAD-binding domain. The FMN semiquinone/hydroquinone redox couple was found to be similar in both constructs. The isolated FAD-binding domain, generated by controlled proteolysis of the reductase domain, was found to have similar FAD reduction potentials to the isolated reductase domain. Both formed a FAD-hydroquinone/NADP(+) charge-transfer complex with a long-wavelength absorption band centered at 780 nm. Formation of this complex resulted in thermodynamic destabilization of the FAD semiquinone relative to the hydroquinone and a 30 mV increase in the FAD semiquinone/hydroquinone reduction potential. Binding of ADP, however, had little effect. The possible role of the nicotinamide/FADH(2) stacking interaction in controlling electron transfer and its likely dependence on protein conformation are discussed.     

The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain

Phe(1395) stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe(1395) was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP(+) binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP(+) with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP(+) dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP(+)-bound state as compared with wild type. We conclude that Phe(1395) regulates nNOSr catalysis in two ways. It facilitates NADP(+) release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer.     

Electron transfer is activated by calmodulin in the flavin domain of human neuronal nitric oxide synthase

The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.     

Chimeric enzymes of cytochrome P450 oxidoreductase and neuronal nitric-oxide synthase reductase domain reveal structural and functional differences

The nitric-oxide synthases (NOSs) are comprised of an oxygenase domain and a reductase domain bisected by a calmodulin (CaM) binding region. The NOS reductase domains share approximately 60% sequence similarity with the cytochrome P450 oxidoreductase (CYPOR), which transfers electrons to microsomal cytochromes P450. The crystal structure of the neuronal NOS (nNOS) connecting/FAD binding subdomains reveals that the structure of the nNOS-connecting subdomain diverges from that of CYPOR, implying different alignments of the flavins in the two enzymes. We created a series of chimeric enzymes between nNOS and CYPOR in which the FMN binding and the connecting/FAD binding subdomains are swapped. A chimera consisting of the nNOS heme domain and FMN binding subdomain and the CYPOR FAD binding subdomain catalyzed significantly increased rates of cytochrome c reduction in the absence of CaM and of NO synthesis in its presence. Cytochrome c reduction by this chimera was inhibited by CaM. Other chimeras consisting of the nNOS heme domain, the CYPOR FMN binding subdomain, and the nNOS FAD binding subdomain with or without the tail region also catalyzed cytochrome c reduction, were not modulated by CaM, and could not transfer electrons into the heme domain. A chimera consisting of the heme domain of nNOS and the reductase domain of CYPOR reduced cytochrome c and ferricyanide at rates 2-fold higher than that of native CYPOR, suggesting that the presence of the heme domain affected electron transfer through the reductase domain. These data demonstrate that the FMN subdomain of CYPOR cannot effectively substitute for that of nNOS, whereas the FAD subdomains are interchangeable. The differences among these chimeras most likely result from alterations in the alignment of the flavins within each enzyme construct.