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.     

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.     

Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH: ubiquinone oxidoreductase (complex I)

Conditions for the reversible dissociation of flavin mononucleotide (FMN) from the membrane-bound mitochondrial NADH:ubiquinone oxidoreductase (complex I) are described. The catalytic activities of the enzyme, i.e. rotenone-insensitive NADH:hexaammineruthenium III reductase and rotenone-sensitive NADH:quinone reductase decline when bovine heart submitochondrial particles are incubated with NADH in the presence of rotenone or cyanide at alkaline pH. FMN protects and fully restores the NADH-induced inactivation whereas riboflavin and flavin adenine dinucleotide do not. The data show that the reduction of complex I significantly weakens the binding of FMN to protein thus resulting in its dissociation when the concentration of holoenzyme is comparable with K(d ( approximately 10(-8)M at pH 10.0).     

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.     

Molecular dissection of human methionine synthase reductase: determination of the flavin redox potentials in full-length enzyme and isolated flavin-binding domains

Human methionine synthase reductase (MSR) catalyzes the NADPH-dependent reductive methylation of methionine synthase. MSR is 78 kDa flavoprotein belonging to a family of diflavin reductases, with cytochrome P450 reductase (CPR) as the prototype. MSR and its individual flavin-binding domains were cloned as GST-tagged fusion proteins for expression and purification from Escherichia coli. The isolated flavin domains of MSR retain UV-visible and secondary structural properties indicative of correctly folded flavoproteins. Anaerobic redox titrations on the individual domains assisted in assignment of the midpoint potentials for the high- and low-potential flavin. For the isolated FMN domain, the midpoint potentials for the oxidized/semiquinone (ox/sq) couple and semiquinone/hydroquinone (sq/hq) couple are -112 and -221 mV, respectively, at pH 7.0 and 25 degrees C. The corresponding couples in the isolated FAD domain are -222 mV (ox/sq) and -288 mV (sq/hq). Both flavins form blue neutral semiquinone species characterized by broad absorption peaks in the long-wavelength region during anaerobic titration with sodium dithionite. In full-length MSR, the values of the FMN couples are -109 mV (ox/sq) and -227 mV (sq/hq), and the corresponding couple values for FAD are -254 mV (ox/sq) and -291 mV (sq/hq). Separation of the MSR flavins does not perturb their thermodynamic properties, as midpoint potentials for all four couples are similar in isolated domains and in full-length MSR. The redox properties of MSR are discussed in relation to other members of the diflavin oxidoreductase family and the mechanism of electron transfer.     

Intraprotein electron transfer in a two-domain construct of neuronal nitric oxide synthase: the output state in nitric oxide formation

Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s(-)(1)) was increased approximately 10-fold compared to that in the absence of CaM (22 s(-)(1)). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.     

Thermodynamics of oxidation-reduction reactions in mammalian nitric-oxide synthase isoforms

The three mammalian nitric-oxide synthases produce NO from arginine in a reaction requiring 3 electrons per NO, which are supplied to the catalytic center from NADPH through reductase domains incorporating FAD and FMN cofactors. The isoforms share a common reaction mechanism and requirements for reducing equivalents but differ in regulation; the endothelial and neuronal isoforms are controlled by calcium/calmodulin modulation of the electron transfer system, while the inducible isoform binds calmodulin at all physiological Ca(2+) concentrations and is always on. The thermodynamics of electron transfer through the flavin domains in all three isoforms are basically similar. The major flavin states are FMN, FMNH., FMNH(2), FAD, FADH., and FADH(2). The FMN/FMNH. couple is high potential ( approximately 100 mV) in all three isoforms and is unlikely to be catalytically competent; the other three flavin couples form a nearly isopotential group clustered around -250 mV. Reduction of the flavins by the pyridine nucleotide couple at -325 mV is thus moderately thermodynamically favorable. The ferri/ferroheme couple in all three isoforms is approximately -270 mV in the presence of saturating arginine. Ca(2+)/calmodulin has no effect on the potentials of any of the couples in endothelial nitric-oxide synthase (eNOS) or neuronal nitric-oxide synthase (nNOS). The pH dependence of the flavin couples suggests the presence of ionizable groups coupled to the flavin redox/protonation states.     

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.     

Mutation of the heme-binding crevice of flavocytochrome b2 from Saccharomyces cerevisiae: altered heme potential and absence of redox cooperativity between heme and FMN centers.

Kinetic and thermodynamic properties of yeast flavocytochrome b2 (EC 1.1.2.3) are modified by the product pyruvate, which binds to the flavosemiquinone (FSQ) form of the prosthetic flavin and decreases the thermodynamic driving force for electron transfer from FSQ to heme. Pyruvate inhibits flavocytochrome b2, but the catalytic competence of pyruvate-ligated FSQ in intramolecular electron transfer to heme is unclear; one kinetic study suggested pyruvate prevented this reaction [Tegoni, M, Janot J.-M., & Labeyrie, F. (1990) Eur. J. Biochem. 190, 329-342], while laser flash photolysis indicated pyruvate was essential [Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555]. To address this problem, wild-type (WT) and mutant (L36I) flavocytochromes b2 have been expressed in Escherichia coli. Both forms incorporated heme and FMN prosthetic groups and were catalytically active. The mutation L36I was a conservative substitution within the heme-binding crevice and was designed to alter the midpoint potential (Em) of the heme to alter the pyruvate-FSQ/heme equilibrium. Potentiometric titrations yielded Em values (pH 7.0, 25 degrees C) of +8 and -28 mV for WT and L36I forms, respectively. The FMN midpoint potentials in the absence of pyruvate (-58 mV, n = 2) were identical within experimental error in WT and L36I species and were also identical (+5 mV, n = 1) in the presence of pyruvate. These results indicated the absence of redox cooperativity between FMN and heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic properties of the redox centers of Na(+)-translocating NADH:quinone oxidoreductase

Redox titration of all optically detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) at pH 7.5 showed that the functionally active enzyme possesses only three titratable flavin cofactors, one noncovalently bound FAD and two covalently bound FMN residues. All three flavins undergo different redox transitions during the function of the enzyme. The noncovalently bound FAD works as a "classical" two-electron carrier with a midpoint potential (E(m)) of -200 mV. Each of the FMN residues is capable of only one-electron reduction: one from neutral flavosemiquinone to fully reduced flavin (E(m) = 20 mV) and the other from oxidized flavin to flavosemiquinone anion (E(m) = -150 mV). The lacking second half of the redox transitions for the FMNs cannot be reached under our experimental conditions and is most likely not employed in the catalytic cycle. Besides the flavins, a [2Fe-2S] cluster was shown to function in the enzyme as a one-electron carrier with an E(m) of -270 mV. The midpoint potentials of all the redox transitions determined in the enzyme were found to be independent of Na(+) concentration. Even the components that exhibit very strong retardation in the rate of their reduction by NADH at low sodium concentrations experienced no change in the E(m) values when the concentration of the coupling ion was changed 1000 times. On the basis of these data, plausible mechanisms for the translocation of transmembrane sodium ions by Na(+)-NQR are discussed.     

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.     

The succinate dehydrogenase assembly factor,SdhE, is required for the flavinylation and activation of fumarate reductase in bacteria

The activity of the respiratory enzyme fumarate reductase (FRD) is dependent on the covalent attachment of the redox cofactor flavin adenine dinucleotide (FAD). We demonstrate that the FAD assembly factor SdhE, which flavinylates and activates the respiratory enzyme succinate dehydrogenase (SDH), is also required for the complete activation and flavinylation of FRD. SdhE interacted with, and flavinylated, the flavoprotein subunit FrdA, whilst mutations in a conserved RGxxE motif impaired the complete flavinylation and activation of FRD. These results are of widespread relevance because SDH and FRD play an important role in cellular energetics and are required for virulence in many important bacterial pathogens.     

Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin.     

Circular dichroism and potentiometry of FAD, heme and Mo-pterin prosthetic groups of assimilatory nitrate reductase

Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric-oxide synthase output state. Design and properties of nitric-oxide synthase oxygenase/FMN domain constructs

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH. radical signals in inducible nitric-oxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquinone couple = +70 mV, FMN semiquinone/hydroquinone couple = -180 mV, and heme = -180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.     

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).     

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.     

Influence of the protein environment on the redox potentials of flavodoxins from Clostridium beijerinckii

The flavin mononucleotide (FMN) quinones in flavodoxin have two characteristic redox potentials, namely, Em(FMNH./FMNH-) for the one-electron reduction of the protonated FMN (E1) and Em(FMN/FMNH.) for the proton-coupled one-electron reduction (E2). These redox potentials in native and mutant flavodoxins obtained from Clostridium beijerinckii were calculated by considering the protonation states of all titratable sites as well as the energy contributed at the pKa value of FMN during protonation at the N5 nitrogen (pKa(N5)). E1 is sensitive to the subtle differences in the protein environments in the proximity of FMN. The protein dielectric volume that prevents the solvation of charged FMN quinones is responsible for the downshift of 130-160 mV of the E1 values with respect to that in an aqueous solution. The influence of the negatively charged 5'-phosphate group of FMN quinone on E1 could result in a maximum shift of 90 mV. A dramatic difference of 130 mV in the calculated E2 values of FMN quinone of the native and G57T mutant flavodoxins is due to the difference in the pKa(N5) values. This is due to the difference in the influence exerted by the carbonyl group of the protein backbone at residue 57.     

The respiratory chain of the thermophilic archaeon Sulfolobus metallicus: studies on the type-II NADH dehydrogenase

The membranes of the thermoacidophilic archaeon Sulfolobus metallicus exhibit an oxygen consumption activity of 0.5 nmol O(2) min(-1) mg(-1), which is insensitive to rotenone, suggesting the presence of a type-II NADH dehydrogenase. Following this observation, the enzyme was purified from solubilised membranes and characterised. The pure protein is a monomer with an apparent molecular mass of 49 kDa, having a high N-terminal amino acid sequence similarity towards other prokaryotic enzymes of the same type. It contains a covalently attached flavin, which was identified as being FMN by 31P-NMR spectroscopy, a novelty among type-II NADH dehydrogenases. Metal analysis showed the absence of iron, indicating that no FeS clusters are present in the protein. The average reduction potential of the FMN group was determined to be +160 mV, at 25 degrees C and pH 6.5, by redox titrations monitored by visible spectroscopy. Catalytically, the enzyme is a NADH:quinone oxidoreductase, as it is capable of transferring electrons from NADH to several quinones, including ubiquinone-1, ubiquinone-2 and caldariella quinone. Maximal turnover rates of 195 micromol NADH oxidized min(-1) mg(-1) at 60 degrees C were obtained using ubiquinone-2 as electron acceptor, after enzyme dilution and incubation with phospholipids.     

Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase

Rat liver microsomal NADPH-cytochrome P-450 reductase was prepared free of detectable amounts of FMN by a new procedure based on the exchange of this flavin into apoflavodoxin. The resulting FMN-free reductase binds NADP in the oxidized state with the same affinity (Kd = 5 microM) and stoichiometry (1:1 molar ratio) as does the native enzyme. Both the native and FMN-free reductase catalyze rapid reduction of ferricyanide, but the ability to reduce th 5,6-benzoflavone-inducible form of the liver microsomal cytochrome P-450 (P-450LM4) is lost upon removal of FMN. The FMN-free enzyme was reconstituted with artificial flavins which, in the free state, have oxidation-reduction potentials ranging from -152 to -290 mV, including 5-carba-5-deaza-FMN and several FMN analogs with a halogen or sulfur substituent on the dimethylbenzene portion of the ring system. Enzyme reconstituted with 5-carba-5-deaza-FMN has catalytic properties which are not significantly different from those of the FMN-free reductase, and is unable to reduce P-450LM4. On the other hand, the ability to reduce P-450LM4 and the other FMN-dependent activities of the native reductase are restored by substitution of several other analogs for FMN, but the kinetics of P-450LM4 reduction, studied under anaerobic conditions by stopped flow spectrophotometry, are significantly altered. The oxidation-reduction behavior of enzyme reconstituted with 7-nor-7-Br-FMN is substantially different from that of the native enzyme, and less thermodynamic stabilization of the semiquinone is observed with this flavin analog. In contrast, the oxidation-reduction properties of enzyme containing 8-nor-8-mercapto-FMN are similar to those of the native enzyme, but the spectral properties are significantly different. As shown in a stopped flow experiment, reduction of this FMN analog precedes reduction of P-450LM4 when a complex of the flavoprotein and P-450LM4 is allowed to react with NADPH. Our experiments support a sequence of electron transfer in this enzyme system as follows: NADPH leads to FAD leads to FMN leads to P-450. We propose that the enzyme cycles between a le- and a 3e-reduced state during turnover and that electrons are donated to acceptors via the reaction, FMNH2 leads to FMNH ..