Ferric reductase activity of the ArsH protein from Acidithiobacillus ferrooxidans

The arsH gene is one of the arsenic resistance system in bacteria and eukaryotes. The ArsH protein was annotated as a NADPH-dependent flavin mononucleotide (FMN) reductase with unknown biological function. Here we report for the first time that the ArsH protein showed high ferric reductase activity. Glu104 was an essential residue for maintaining the stability of the FMN cofactor. The ArsH protein may perform an important role for cytosolic ferric iron assimilation in vivo.     

Crystal structure of an apo form of Shigella flexneri ArsH protein with an NADPH-dependent FMN reductase activity

The arsH gene or its homologs are a frequent part of the arsenic resistance system in bacteria and eukaryotes. Although a specific biological function of the gene product is unknown, the ArsH protein was annotated as a member of the NADPH-dependent FMN reductase family based on a conserved (T/S)XRXXSX(T/S) fingerprint motif common for FMN binding proteins. Presented here are the first crystal structure of an ArsH protein from Shigella flexneri refined at 1.7 A resolution and results of enzymatic activity assays that revealed a strong NADPH-dependent FMN reductase and low azoreductase activities. The ArsH apo protein has an alpha/beta/alpha-fold typical for FMN binding proteins. The asymmetric unit consists of four monomers, which form a tetramer. Buried surface analysis suggests that this tetramer is likely to be the relevant biological assembly. Dynamic light scattering experiments are consistent with this hypothesis and show that ArsH in solution at room temperature does exist predominantly in the tetrameric form.     

The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.     

Structure-function relations in flavodoxins

Summary Flavodoxins are low molecular weight, FMN containing, proteins which function as electron transfer agents in a variety of microbial metabolic processes, including nitrogen fixation. Utilizing structural information obtained from x-ray crystal analysis, it has been possible to derive some new and important insights into the relationships which exist between flavin properties and protein environment by comparing the spectroscopic, thermodynamic and kinetic behavior of the flavodoxins with that of free flavin. Thus, for example, a qualitative understanding of the contribution of the protein to flavin redox potentials, semiquinone reactivity and mechanism of electron transfer is beginning to emerge. The highly negative redox potential required for the biochemical activity of the flavodoxins is accomplished by stabilizing the semiquinone via a hydrogen bond to the N-5 position of the flavin and destabilizing the fully-reduced form by constraining it to assume an unfavorable planar conformation. The reactivity of the semiquinone form is lowered by the aforementioned hydrogen bond, as well as by an interaction with a tryptophan residue in the binding site. Electron transfer is accomplished through the exposed dimethylbenzene ring of the bound coenzyme. Although it is not possible at present to determine the extent to which this understanding can be generalized to other flavoproteins, it is clear that a study of the flavodoxins will provide us with at least some of the principles which biological systems have used to modify flavin properties to fulfill a biochemical need.     

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.     

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.     

Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase

Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chem. Soc. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.     

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.     

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.     

Kinetic and thermodynamic characterization of the common polymorphic variants of human methionine synthase reductase

Human methionine synthase reductase (MSR) is a protein containing both FAD and FMN, and it reactivates methionine synthase that has lost activity due to oxidation of cob(I)alamin to cob(II)alamin. In this study, anaerobic redox titrations were employed to determine the midpoint reduction potentials for the flavin cofactors in two highly prevalent polymorphic variants of MSR, I22/L175 and M22/S175. The latter is a genetic determinant of plasma homocysteine levels and has been linked to premature coronary artery disease, Down's syndrome, and neural tube defects. The I22/L175 polymorphism has been described in a homocystinuric patient. Interestingly, this polymorphism is in the extended linker region between the two flavin domains, which may mediate or facilitate interaction with methionine synthase. In MSR I22/L175, the FMN potentials are -103 mV (oxidized/semiquinone) and -175 mV (semiquinone/hydroquinone) at pH 7.0 and 25 degrees C, and the corresponding FAD potentials are -252 and -285 mV, respectively. For the M22/S175 variants, the values of the four midpoint potentials are -114 mV (FMN oxidized/semiquinone), -212 mV (FMN semiquinone/hydroquinone), -236 mV (FAD oxidized/semiquinone), and -264 mV (FAD semiquinone/hydroquinone). The midpoint potential values in the two variants are generally comparable to those originally determined for the MSR I22/S175 variant [Wolthers, K. R. (2003) Biochemistry 42, 3911-3920], with relatively minor variations in the different redox couples. In each case, blue neutral flavin semiquinone species are stabilized on both flavins, and are characterized by a broad absorption band in the long wavelength region. In addition, stopped-flow absorption and fluorescence spectroscopy were used to study the pre-steady state reduction kinetics by NADPH of the two polymorphic variants. The reversible kinetic model proposed for wild-type MSR was validated for the I22/L175 and M22/S175 variants. Thus, the biochemical penalties associated with these polymorphisms, which result in less effective methionine synthase activation, do not appear to result from differences in their reduction kinetics. It is likely that differences in their relative affinities for the redox partner, methionine synthase, underlie the differences in the relative efficiencies of reductive activation exhibited by the variants.     

Transient kinetics of redox reactions of flavodoxin: effects of chemical modification of the flavin mononucleotide prosthetic group on the dynamics of intermediate complex formation and electron transfer

The effects of structural modifications of the flavin mononucleotide (FMN) prosthetic group of Clostridium pasteurianum flavodoxin on the kinetics of electron transfer to the oxidized form (from 5-deazariboflavin semiquinone produced by laser flash photolysis) and from the semiquinone form (to horse heart cytochrome c by using stopped-flow spectrophotometry) have been investigated. The analogues used were 7,8-dichloro-FMN, 8-chloro-FMN, 7-chloro-FMN, and 5,6,7,8-tetrahydro-FMN. The ionic strength dependence of cytochrome c reduction was not affected by chlorine substitution, although the specific rate constants for complex formation and decay were appreciably smaller. On the other hand, all of the chlorine analogues had the same rate constant for deazariboflavin semiquinone oxidation. The rate constants for tetrahydro-FMN flavodoxin semiquinone reduction of cytochrome c were considerably smaller than those for the native protein. The implications of these results for the electron-transfer mechanism of flavodoxin are discussed.     

Potentiometric and further kinetic characterization of the flavin-binding domain of Saccharomyces cerevisiae flavocytochrome b2. Inhibition by anions binding in the active site

Saccharomyces cerevisiae flavocytochrome b2 (L-lactate:cytochrome c oxido reductase, EC 1.1.2.3) is a homotetramer, with FMN and protoheme IX binding on separate domains. The flavin-binding domains form the enzyme tetrameric core, while the cytochrome b2 domains appear to be mobile around a hinge region (Xia, Z. X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 867-863). The enzyme catalyzes electron transfer from L-lactate to cytochrome c, or to nonphysiological acceptors such as ferricyanide, via FMN and heme b2. The kinetics of this multistep reaction are complex. In order to clarify some of its aspects, the tetrameric FMN-binding domain (FDH domain) has been independently expressed in Escherichia coli (Balme, A., Brunt, C. E., Pallister, R., Chapman, S. K., and Reid, G. A. (1995) Biochem. J. 309, 601-605). We present here an additional characterization of this domain. In our hands, it has essentially the same ferricyanide reductase activity as the holo-enzyme. The comparison of the steady-state kinetics with ferricyanide as acceptor and of the pre-steady-state kinetics of flavin reduction, as well as the kinetic isotope effects of the reactions using L-2-[2H]lactate, indicates that flavin reduction is the limiting step in lactate oxidation. During the oxidation of the reduced FDH domain by ferricyanide, the oxidation of the semiquinone is much faster than the oxidation of two-electron-reduced flavin. This order of reactivity is reversed during flavin to heme b2 transfer in the holo-enzyme. Potentiometric studies of the protein yielded a standard redox potential for FMN at pH 7.0, E(o)7, of -81 mV, a value practically identical to the published value of -90 mV for FMN in holo-flavocytochrome b2. However, as expected from the kinetics of the oxidative half-reaction, the FDH domain was characterized by a significantly destabilized flavin semiquinone state compared with holo-enzyme, with a semiquinone formation constant K of 1.25-0.64 vs 33.5, respectively (Tegoni, M., Silvestrini, M. C., Guigliarelli, B., Asso, M., and Bertrand, P. (1998) Biochemistry, 37, 12761-12771). As in the holo-enzyme, the semiquinone state in the FDH domain is significantly stabilized by the reaction product, pyruvate. We also studied the inhibition exerted in the steady and pre steady states by the reaction product pyruvate and by anions (bromide, chloride, phosphate, acetate), with respect to both flavin reduction and reoxidation. The results indicate that these compounds bind to the oxidized and the two-electron-reduced forms of the FDH domain, and that excess L-lactate also binds to the two-electron-reduced form. These findings point to the existence of a common or strongly overlapping binding site. A comparison of the effect of the anions on WT and R289K holo-flavocytochromes b2 indicates that invariant R289 belongs to this site. According to literature data, it must also be present in other members of the family of L-2-hydroxy acid-oxidizing enzymes.     

FAD semiquinone stability regulates single- and two-electron reduction of quinones by Anabaena PCC7119 ferredoxin:NADP+ reductase and its Glu301Ala mutant

Flavoenzymes may reduce quinones in a single-electron, mixed single- and two-electron, and two-electron way. The mechanisms of two-electron reduction of quinones are insufficiently understood. To get an insight into the role of flavin semiquinone stability in the regulation of single- vs. two-electron reduction of quinones, we studied the reactions of wild type Anabaena ferredoxin:NADP(+)reductase (FNR) with 48% FAD semiquinone (FADH*) stabilized at the equilibrium (pH 7.0), and its Glu301Ala mutant (8% FADH* at the equilibrium). We found that Glu301Ala substitution does not change the quinone substrate specificity of FNR. However, it confers the mixed single- and two-electron mechanism of quinone reduction (50% single-electron flux), whereas the wild type FNR reduces quinones in a single-electron way. During the oxidation of fully reduced wild type FNR by tetramethyl-1,4-benzoquinone, the first electron transfer (formation of FADH*) is about 40 times faster than the second one (oxidation of FADH*). In contrast, the first and second electron transfer proceeded at similar rates in Glu301Ala FNR. Thus, the change in the quinone reduction mechanism may be explained by the relative increase in the rate of second electron transfer. This enabled us to propose the unified scheme of single-, two- and mixed single- and two-electron reduction of quinones by flavoenzymes with the central role of the stability of flavin/quinone ion-radical pair.     

A Switch between One- and Two-electron Chemistry of the Human Flavoprotein Iodotyrosine Deiodinase Is Controlled by Substrate

Reductive dehalogenation is not typical of aerobic organisms but plays a significant role in iodide homeostasis and thyroid activity. The flavoprotein iodotyrosine deiodinase (IYD) is responsible for iodide salvage by reductive deiodination of the iodotyrosine derivatives formed as byproducts of thyroid hormone biosynthesis. Heterologous expression of the human enzyme lacking its N-terminal membrane anchor has allowed for physical and biochemical studies to identify the role of substrate in controlling the active site geometry and flavin chemistry. Crystal structures of human IYD and its complex with 3-iodo-l-tyrosine illustrate the ability of the substrate to provide multiple interactions with the isoalloxazine system of FMN that are usually provided by protein side chains. Ligand binding acts to template the active site geometry and significantly stabilize the one-electron-reduced semiquinone form of FMN. The neutral form of this semiquinone is observed during reductive titration of IYD in the presence of the substrate analog 3-fluoro-l-tyrosine. In the absence of an active site ligand, only the oxidized and two-electron-reduced forms of FMN are detected. The pH dependence of IYD binding and turnover also supports the importance of direct coordination between substrate and FMN for productive catalysis.     

Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H:nitroreductase: quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes two-electron reduction of a series of quinoidal compounds according to a "ping-pong" scheme, with marked substrate inhibition by quinones. The steady-state catalytic constants (k(cat)) range from 0.1 to 1600s(-1), and bimolecular rate constants (k(cat)/K(m)) range from 10(3) to 10(8)M(-1)s(-1). Quinones, nitroaromatic compounds and competitive to NADH inhibitor dicumarol, quench the flavin mononucleotide (FMN) fluorescence of nitroreductase. The reactivity of NR with single-electron acceptors is consistent with an "outer-sphere" electron transfer model, taking into account high potential of FMN semiquinone/FMNH(-) couple and good solvent accessibility of FMN. However, the single-electron acceptor 1,1(')-dibenzyl-4,4(')-bipyridinium was far less reactive than quinones possessing similar single-electron reduction potentials (E(1)(7)). For all quinoidal compounds except 2-hydroxy-1,4-naphthoquinones, there existed parabolic correlations between the log of rate constants of quinone reduction and their E(1)(7) or hydride-transfer potential (E(7)(Q/QH(-))). Based on pH dependence of rate constants, a single-step hydride transfer seems to be a more feasible quinone reduction mechanism. The reactivities of 2-hydroxy-1,4-naphthoquinones were much higher than expected from their reduction potential. Most probably, their enhanced reactivity was determined by their binding at or close to the binding site of NADH and dicumarol, whereas other quinones used the alternative, currently unidentified binding site.     

Studies with substrate and cofactor analogues provide evidence for a radical mechanism in the chorismate synthase reaction

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC(50) of the fluorinated substrate analogue was 0.5 and 250 microm with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.     

Calmodulin activates intramolecular electron transfer between the two flavins of neuronal nitric oxide synthase flavin domain

The neuronal NO synthase (nNOS) flavin domain, which has similar redox properties to those of NADPH-cytochrome P450 reductase (P450R), contains binding sites for calmodulin, FAD, FMN, and NADPH. The aim of this study is to elucidate the mechanism of activation of the flavin domain by calcium/calmodulin (Ca(2+)/CaM). In this study, we used the recombinant nNOS flavin domains, which include or delete the calmodulin (CaM)-binding site. The air-stable semiquinone of the nNOS flavin domains showed similar redox properties to the corresponding FAD-FMNH(&z.ccirf;) of P450R. In the absence or presence of Ca(2+)/CaM, the rates of reduction of an FAD-FMN pair by NADPH have been investigated at different wavelengths, 457, 504 and 590 nm by using a stopped-flow technique and a rapid scan spectrophotometry. The reduction of the oxidized enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron equivalent mechanisms, and the formation of semiquinone (increase of absorbance at 590 nm) was significantly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form of the enzyme was also rapidly reduced by NADPH. The results suggest that an intramolecular one-electron transfer between the two flavins is activated by the binding of Ca(2+)/CaM. The F(1)H(2), which is the fully reduced form of the air-stable semiquinone, can donate one electron to the electron acceptor, cytochrome c. The proposed mechanism of activation by Ca(2+)/CaM complex is discussed on the basis of that provided by P450R.     

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.     

Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp. PCC 7119.

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.     

Laser flash photolysis study of intermolecular and intramolecular electron transfer in trimethylamine dehydrogenase

Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)