Eur J Biochem 271, 2548–2560 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04185.x Thermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395 Adrian J Dunford, Ker R Marshall, Andrew W Munro and Nigel S Scrutton Department of Biochemistry, University of Leicester, UK In rat neuronal nitric oxide synthase, Phe1395 is positioned over the FAD isoalloxazine ring This is replaced by Trp676 in human cytochrome P450 reductase, a tryptophan in related diflavin reductases (e.g methionine synthase reductase and novel reductase 1), and tyrosine in plant ferredoxinNADP+ reductase Trp676 in human cytochrome P450 reductase is conformationally mobile, and plays a key role in enzyme reduction Mutagenesis of Trp676 to alanine results in a functional NADH-dependent reductase Herein, we describe studies of rat neuronal nitric oxide synthase FAD domains, in which the aromatic shielding residue Phe1395 is replaced by tryptophan, alanine and serine In steady-state assays the F1395A and F1395S domains have a greater preference for NADH compared with F1395W and wildtype Stopped-flow studies indicate flavin reduction by NADH is significantly faster with F1395S and F1395A domains, suggesting that this contributes to altered preference in coenzyme specificity Unlike cytochrome P450 reductase, the switch in coenzyme specificity is not attributed to differential binding of NADPH and NADH, but probably results from improved geometry for hydride transfer in the F1395S– and F1395A–NADH complexes Potentiometry indicates that the substitutions not significantly perturb thermodynamic properties of the FAD, although considerable changes in electronic absorption properties are observed in oxidized F1395A and F1395S, consistent with changes in hydrophobicity of the flavin environment In wild-type and F1395W FAD domains, prolonged incubation with NADPH results in development of the neutral blue semiquinone FAD species This reaction is suppressed in the mutant FAD domains lacking the shielding aromatic residue The nitric oxide synthases (NOS) catalyse the NADPH- and oxygen-dependent conversion of L-arginine to L-citrulline and nitric oxide (NO) [1–3] They are dimeric flavohaem enzymes and each monomer comprises a C-terminal diflavin reductase domain and an N-terminal oxygenase domain [4–7] The reductase domain is related structurally and functionally to cytochrome P450 reductase (CPR) [8,9], methionine synthase reductase (MSR [10]); and the cancerassociated protein NR1 [11] The N-terminal oxygenase domain of NOS contains one mole equivalent of haem and possesses binding sites for L-arginine and (6R)-5,6,7,8tetrahydrobiopterin [12] The reductase and oxygenase domains are linked by a calmodulin (CaM) binding sequence [8,13–15], and CaM acts by releasing an NADPH-dependent conformational lock [16] CaM binding has been proposed to enhance the rate of interflavin electron transfer [17–19], although this remains a controversial aspect of CaM regulation of electron transfer [20] Enhanced steady-state rates of cytochrome c reduction by NOS reductase by CaM binding are, in the main, attributed to faster FMN to cytochrome c electron transfer rates in the presence of CaM, through release of the NADPH-dependent conformational lock [16] Of the three NOS isoforms, the inducible NOS isoform is expressed with CaM tightly bound [14] and regulation of activity is primarily through transcriptional processes; the activities of endothelial NOS and neuronal NOS (nNOS) are regulated by CaM binding, which in turn is controlled by intracellular calcium levels [4,5,7] and is mediated by an autoinhibitory sequence in the FMN domain [21] NADPH is the preferred reducing coenzyme for nNOS and the other NOS isoforms and this property is shared by other members of the diflavin reductase family of enzymes, including P450 BM3 [22], CPR [23], MSR [24] and NR1 [11] The structure of the NOS FAD domain indicates that Phe1395 stacks over the FAD isoalloxazine ring [25] This residue is equivalent to Trp677 in rat CPR which likewise stacks over the FAD isoalloxazine ring [9] On binding NADPH, this residue must move to allow hydride transfer from NADPH to FAD That this residue is mobile has been confirmed by stopped-flow kinetic analysis of FAD reduction with wild-type human CPR and the W676H mutant (equivalent to W677 in rat CPR) [26,27] W676 facilitates Correspondence to A W Munro and N S Scrutton, Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7HR, UK Fax: + 44 116252 3369, Tel.: + 44 116223 1337 or + 44 116252 3464, E-mail: awm9@le.ac.uk or nss4@le.ac.uk Abbreviations: NOS, nitric oxide synthase; CPR, cytochrome P450 reductase; MSR, methionine synthase reductase; CaM, calmodulin; BM3, Bacillus megaterium flavocytochrome P450 (Received 25 February 2004, revised 22 April 2004, accepted 26 April 2004) Keywords: coenzyme specificity; cytochrome P450 reductase; electron transfer; nitric oxide synthase; redox potential Ó FEBS 2004 Kinetic and thermodynamic properties of nNOS FAD domains (Eur J Biochem 271) 2549 release of NADP+ following hydride transfer, a process that is impaired in the W676H mutant enzyme [27] Moreover, the coenzyme specificity of human CPR is switched in favour of NADH in the W676A enzyme, indicating a role for W676 in coenzyme discrimination [28] A similar switch in coenzyme specificity has been reported for the F1395S mutant of nNOS [29] suggesting that Phe1395 in nNOS plays a role similar to that of W676 in human CPR as regards coenzyme binding Similar studies on Y308 mutants of the pea choloroplast ferredoxin reductase enzyme indicated large changes in pyridine nucleotide selectivity towards NADH in the Y308G and Y308S mutants The absence of the tyrosine was conjectured to stabilize interaction with the nicotinamide group common to both NADH and NADPH [30] In rat nNOS, residue Phe1395 is located close to the re face of the FAD isoalloxazine ring [25], and is structurally equivalent to Trp677 in rat CPR (Fig 1A) [9,47] An aromatic residue is found in all sequence-related mammalian diflavin reductases sequenced to date, and also in plant ferredoxin reductases that are structurally similar to the FAD domains of NOS and CPR (Fig 1B) In an attempt to understand in detail the role of Phe1395 in the catalytic mechanism of nNOS, we have isolated wild-type and mutant forms of the nNOS FAD domain in which Phe1395 was exchanged for a serine (F1395S), alanine (F1395A) and tryptophan (F1395W) residue The value of studying the thermodynamic and kinetic properties of individual Fig The coenzyme-binding site in nNOS FAD domain and sequence alignments around the conserved aromatic residue in this domain (A) The nicotinamide-binding site of nNOS FAD domain showing the position of Phe1395 in relation to the structurally equivalent W677 in rat CPR The FAD of CPR (PDB code 1AMO) and rat NOS (PDB code 1F2O) are shown in yellow NADP+ in the ÔoffÕ conformation (PDB code 1JA1) is shown in blue, and in the ÔonÕ conformation (PDB code 1J9Z) in green (see [48] for details) Phe1395 (rat nNOS) is shown in purple; Trp677 (rat CPR) is shown in pink (B) Alignment of sequences for mammalian diflavin reductases and plant ferredoxin reductase in the region of the conserved aromatic residues that shield the FAD isoalloxazine ring nNOS, rat neuronal nitric oxide synthase; eNOS, rat endothelial nitric oxide synthase; iNOS, rat inducible nitric oxide synthase; CPR, human cytochrome P450 reductase; NR1, human novel oxidoreductase 1; MSR, human methionine synthase reductase; BM3, Bacillus megaterium flavocytochrome P450 BM3; FNR, spinach ferredoxin NADP+ reductase The relevant flavin shielding aromatic residue is underlined in bold text in all cases domains of complex multidomain enzymes has been demonstrated in previous work with P450 BM3 [31], rat nNOS [32], MSR [33,34], human NR1 [35] and human CPR [26,36] Clear evidence for the multidomain nature of these enzymes has been obtained through the stable expression of domains that bind their cognate cofactors and retain catalytic properties typical of the parental diflavin reductases and flavocytochromes (e.g [35,37]) In particular, domain dissection has facilitated precise determination of the midpoint reduction potentials of flavin cofactors, and also of the haems in the case of NOS and P450 BM3 In spectroelectrochemical titrations of the isolated domains, the lack of overlapping spectral contributions from other cofactors present in the full-length enzymes has enabled: (a) deconvolution of the contributions of individual flavin cofactors to the overall absorption changes observed in the intact enzymes; (b) determination of the relative tendencies of individual flavin cofactors to stabilize semiquinone intermediates; and (c) precise determination of the reduction potentials of the one- and two-electron redox couples associated with each flavin (e.g [32,33,36,38]) Studies of individual domains have subsequently assisted in assignment and determination of mid-point reduction potentials for each redox couple in full-length enzymes Stuehr and coworkers have reported that exchange of Phe1395 for serine in full-length rat NOS improves activity with NADH They propose that Phe1395 forms part of a conformational ƠtriggerÕ mechanism that positively or Ĩ FEBS 2004 2550 A J Dunford et al (Eur J Biochem 271) negatively regulates NO synthesis depending on whether CaM is bound [29] In this paper, we have extended the studies of Stuehr and coworkers by investigating the effects of exchanging Phe1395 with serine, alanine and tryptophan in the isolated FAD domain of rat nNOS (F1395W, F1395S and F1395A) and by probing the thermodynamic and kinetic consequences of these mutations We have studied the thermodynamic and kinetic properties of the isolated FAD domain of rat nNOS and three mutant forms The isolated FAD domains have enabled: (a) potentiometric analysis of the wild-type and mutant proteins to probe any thermodynamic consequences of mutation; and (b) studies of the kinetics of flavin reduction by reducing coenzymes in the absence of spectral change arising from electron transfer to the FMN domain Clear differences in steady-state kinetic properties are observed in the mutants, along with a considerable shift in pyridine nucleotide specificity towards NADH for the F1395S and F1395A proteins However, steady-state kinetic differences are not attributed to gross changes in the flavin redox potentials, although effects on the kinetics of FAD reduction are observed Our data are discussed in the light of results from mutagenic studies of related enzymes (particularly CPR), and indicate that there are subtle differences in the roles of the stacking aromatic residues in the different diflavin reductase enzymes and in how they regulate pyridine nucleotide coenzyme specificity and enzymatic properties Experimental procedures Cloning of rat nNOS FAD/NADPH domain The rat nNOS FAD/NADPH domain, amino acid residues 987–1463, was amplified from plasmid pCRNNR [39] comprising a pKK223-3 clone of the rat nNOS reductase domain PCR amplification was performed using Pfu Turbo DNA polymerase (Stratagene) and the forward primer 5¢-GCAATCATATGAGCTGGAAGAGGAACAAGTT CCG-3¢ and the reverse primer 5¢-GGATCCTTAGGA GCTGAAAACCTCATCTGCG-3¢, containing NdeI and BamHI restriction sites, respectively The resultant fragment was gel-purified (QIAquick gel extraction kit, Qiagen) and then A-tailed using Taq DNA polymerase prior to being cloned into pGEM-T Easy (Promega) Clones were verified by automated DNA sequencing prior to being subcloned into NdeI- and BamHI-cut expression vector, pET11a Site-directed mutagenesis of rat nNOS FAD/NADPH domain Residue F1395 of the rat nNOS FAD/NADPH domain was mutated to either A1395, S1395 or W1395 using the nonstrand-displacing DNA polymerase Pfu Turbo and the following mutagenic primer combinations: F1395A, forward primer 5¢-CACGAGGATATCGCTGGAGTCAC CCTC-3¢ and the reverse complement thereof; F1395S, forward primer 5¢-CACGAGGATATCTCTGGAGTCA CCCTCAG-3¢ and its reverse complement; F1395W, 5¢-CCGGTACCACGAGGATATCTGGGGAG-3¢ together with the reverse complementary primer All primers incorporated silent mutations to introduce an EcoRV restriction site (underlined) to assist in mutant screening Mutated bases are given in bold type Cycling parameters for mutagenesis reactions were 95 °C for 30 s followed by 16 cycles of 95 °C for 30 s, 55 °C for and 68 °C for Nonmutated template DNA was then removed by DpnI digestion and mutant DNA transformed into Escherichia coli JM109 Selected clones were first assessed by EcoRV digestion and then verified by automated DNA sequencing Purification of the isolated FAD domains Transformed cells were grown in Terrific Broth [40] Expression of the isolated FAD-domains was induced by addition of isopropyl thio-b-D-galactoside (1 mM) at a culture optical density of 0.8 at 600 nm; cells were grown for a further 24 h at 30 °C Harvested cells were resuspended in lysis buffer [50 mL; 50 mM Tris/HCl pH 7.4 containing 10% (v/v) glycerol, mM CaCl2 and a CompleteTM EDTAfree protease inhibitor tablet (Roche)] Cells were disrupted by sonication, the cell extract clarified by centrifugation (15 000 g, 50 min) and fractionated with ammonium sulfate (FAD domain was recovered in the 30–50% saturation fraction) Enzyme was dialysed exhaustively against lysis buffer, and applied to an anion exchange resin (DE-52) previously equilibrated with lysis buffer The column was washed with lysis buffer (500 mL) and FAD domain was recovered by developing the column with a gradient (0–0.5 M) of KCl Fractions containing FAD domain were pooled, and applied to an affinity resin (2¢5¢-ADP Sepharose) equilibrated with lysis buffer containing 100 mM NaCl After washing ( 250 mL lysis buffer 100 mM NaCl and then  250 mL lysis buffer, 250 mM NaCl), FAD domain was recovered by the application of lysis buffer containing 500 mM NaCl Enzyme was dialysed exhaustively against lysis buffer and stored at )20 °C in the presence of 20% (v/v) glycerol Potentiometry Redox titrations for the nNOS FAD domains (wild-type, F1395A, F1395S and F1395W) were performed in a Belle Technology glove box under a nitrogen atmosphere, essentially as described previously [36] All solutions were degassed under vacuum with argon Oxygen levels were maintained at < p.p.m The protein solution [ 50 lM in mL 100 mM potassium phosphate pH 7.0 in the presence and absence of 10% (v/v) glycerol] was titrated electrochemically according to the method of Dutton [41] using sodium dithionite as reductant and potassium ferricyanide as oxidant Mediators (2 lM phenazine methosulfate, lM 2-hydroxy-1, 4-naphthoquinone, 0.5 lM methyl viologen, and lM benzyl viologen) were included to mediate in the range between +100 and )480 mV as described previously [35,41] At least 15 was allowed to elapse between each addition of reductant/oxidant to allow stabilization of the electrode Spectra (300–800 nm) were recorded using a Cary UV-50 Bio UV-Visible scanning spectrophotometer, using a fibre optic probe immersed in the protein solutions and connected externally to the spectrophotometer The electrochemical potential of the solution was measured using a Hanna pH 211 meter coupled to a Pt/Calomel electrode (Thermo Russell Ltd) at 25 °C The electrode was calibrated Ó FEBS 2004 Kinetic and thermodynamic properties of nNOS FAD domains (Eur J Biochem 271) 2551 using the Fe3+/Fe2+ EDTA couple as a standard (+108 mV) A factor of +244 mV was used to correct relative to the standard hydrogen electrode Spectral data were imported into ORIGIN (Microcal) and spectral subtractions performed to correct for baseline drift during the titrations (bringing absorption back to zero at 800 nm, where there is no significant absorption from the cofactor in oxidized or reduced states) Spectral data were fitted to appropriate Nernst functions in ORIGIN to derive the relevant midpoint reduction potentials of the flavins, as described previously [36] Stopped-flow kinetic measurements Stopped-flow studies were performed using an Applied Photophysics SX.18 MX stopped-flow spectrophotometer contained within an anaerobic glove box (Belle Technology) Measurements were carried out at 25 °C in 50 mM Tris/HCl pH 7.4 containing 10% (v/v) glycerol Protein concentration was lM (reaction cell concentration) for single wavelength work and 10 lM for photodiode array studies All buffers were made oxygen-free by evacuation and extensive bubbling with argon before use Buffers were then placed in the glove box overnight before use Prior to stopped-flow studies, protein samples were treated with potassium hexacyanoferrate, and excess cyanoferrate was removed by rapid gel filtration [Sephadex G25 equilibrated in 50 mM Tris/HCl pH 7.4, 10% (v/v) glycerol] Stopped-flow, multiple-wavelength absorption studies were carried out using a photodiode array detector and X-SCAN software (Applied Photophysics Ltd) Spectral deconvolution was performed by global analysis and numerical integration methods using PROKIN software (Applied Photophysics Ltd) In single wavelength studies, flavin reduction by NADPH was observed at 458 nm Stopped-flow fluorescence experiments used excitation wavelengths of 340 nm (NADPH) and 295 nm (tryptophan) Emission bands were selected using the appropriate band pass filter Results Domain isolation and mutagenesis of Phe1395 Each of the F1395S/A/W FAD domains were purified in high yield (typically 15 mg from L recombinant cells) and in pure form as judged by SDS/PAGE The spectral properties of each are shown in Fig Major alterations in electronic absorption spectra are apparent for the F1395A and F1395S FAD domains In these two mutants, the shorter wavelength absorption band of the FAD is intensified and blue-shifted with respect to the wild-type The F1395W spectrum is virtually indistinguishable from that for wild-type, suggesting a conservative effect of the aromatic replacement on the flavin electronic properties Flavin spectral maxima are located at 457 nm and 398 nm for wild-type and F1395W mutants, with a pronounced shoulder on the longer wavelength band at  480 nm Similarly, the electronic absorption spectra of F1395A and F1395S FAD domains are strongly similar to one another, but distinct from those of wild-type and F1395W proteins Absorption maxima are at 456.5 nm and 387.5 nm for F1395A/S There is a marked increase in the relative intensity of the shorter wavelength band in the F1395A/S mutants, and the shoulder on the longer wavelength band is also much less pronounced than in wild-type and F1395W (Fig 2) The spectral perturbations observed as a consequence of the nonaromatic amino acid substitutions in F1395A/S are consistent with a less hydrophobic environment for the FAD isoalloxazine ring compared to wild-type and F1395W Steady-state kinetic analysis of wild-type and mutant FAD domains Steady-state analysis of nNOS FAD domain-dependent ferricyanide reduction indicates that there are only moderate effects on Km for NADPH induced by the replacement of Steady-state enzyme assays The steady-state activities of wild-type and mutant enzymes using ferricyanide as electron acceptor were determined using a Jasco V-550 UV/visible double-beam spectrophotometer Assays were performed in the double-beam spectrophotometer to take account of any nonenzymemediated reduction of electron acceptor The reference cuvette contained the same mix as the sample cuvette, but the same volume of buffer replaced the enzyme The reaction was initiated by the simultaneous addition of NAD(P)H to both cuvettes Potassium ferricyanide reduction was monitored at 420 nm (De420nm (red-ox) ẳ 1020 M)1ặcm)1) Reactions were performed in 50 mM Tris/HCl pH 7.4, 10% glycerol at 25 °C With ferricyanide as exogenous electron acceptor, saturating concentrations of NADPH (500 lM) were used to determine the Km for the substrate The Km for NAD(P)H was determined at a fixed, saturating concentration of ferricyanide (2 mM) ORIGIN software (Microcal) was used in data fitting to derive Km and kcat values from steady-state assays Fig UV-Visible absorption spectra for wild-type and mutant forms of nNOS FAD domain (all  lM) Protein samples were contained in 50 mM Tris/HCl buffer, 10% (v/v) glycerol, pH 7.4, at 25 °C Spectra are F1395S (dashed line), F1395A (dotted line), WT (solid, thick line) and F1395W (solid, thin line) Ó FEBS 2004 2552 A J Dunford et al (Eur J Biochem 271) Table Steady-state kinetic parameters for ferricyanide reduction by wild-type and mutant forms of the nNOS FAD domain Kinetic parameters were determined using both NADPH and NADH as electron donors The Km and kcat values for ferricyanide (Fe[(CN)6]3–) were determined at a fixed and saturating concentration of NADPH (500 lM) The Km and kcat values for NADPH and NADH were determined at a fixed and saturating concentration of ferricyanide (2 mM) All reactions were performed in 50 mM Tris/HCl, 10% glycerol, pH 7.4, at 25 °C NADPH NADH kcat/Km (lM)1Ỉs)1) )1 nNOS Km (lM) kcat (s ) WT F1395W F1395A F1395S 28.2 15.4 83.5 22.9 161.6 113.5 209.8 50.8 ± ± ± ± 4.5 11.9 4.2 ± ± ± ± 7.2 10.1 6.6 2.4 Km (lM) 5.7 7.4 2.5 2.2 5890 3550 3250 1830 the FAD-stacking phenylalanine with either aromatic (tryptophan) or nonaromatic (alanine, serine) sidechains (Table 1) There is an apparent small decrease in KM for NADPH in the F1395W mutant (Km ¼ 15.4 lM cf 28.2 lM for wild-type) and a larger increase for the F1395A mutant (Km ¼ 83.5 lM) The value for the F1395S mutant is within error of that for the wild-type nNOS FAD domain (Table 1) There are also effects on the kcat values for ferricyanide reduction following mutation, with diminution in kcat for the F1395W and F1395S mutants compared to wild-type (70% and 31%, respectively, of wild-type kcat); interestingly, the kcat for F1395A is increased by 30% over wild-type The net effects on the second order rate constant (kcat/Km) describing the overall efficiency of NADPHdependent ferricyanide reduction is that the F1395W mutant shows a modest improvement (30%) over wildtype, while the nonaromatic substituted mutants are decreased to 44% (F1395A) and 39% (F1395S) of the wild-type value (Table 1) Much more marked effects are seen in NADH-dependent catalysis The apparent Km values are lower in all mutants than in wild-type, with F1395S showing the greatest improvement (Km ¼ 1830 lM compared with 5890 lM for wild-type) While the F1395W mutant has a diminished kcat value (78%) compared to wild-type, both of the F1395A/S mutants show considerable improvements in kcat values (5.9-fold and 2.6-fold, respectively) This leads to even greater improvements in the kcat/Km ratio for the F1395A/S mutants over wild-type (10.7/8.4-fold), with a minor improvement also observed for the F1395W mutant (1.3fold) (Table 1) The large increases in catalytic efficiency of the nonaromatic substituted mutants mirror the results observed in earlier studies with the human CPR [28] The increases in efficiency of ferricyanide reduction are also consistent with the results of Stuehr and coworkers in their analysis of the effects of the F1395S mutation in full-length CaM-bound nNOS [29] For full-length F1395S nNOS, a  30-fold increase over wild-type was obtained for the the apparent kcat for NADH-dependent ferricyanide reduction The rather smaller enhancement of kcat reported here for the isolated FAD domain F1395S mutant may represent a more accurate representation of the effect of the mutation on electron transfer from NADH through to ferricyanide, since effects of CaM (particularly in view of the interplay between the F1395 sidechain and CaM indicated by the studies of Adak et al [29]) and interflavin electron transfer on the steady-state kinetics can be ruled out Steady-state ± ± ± ± kcat/Km (lM)1Ỉs)1) )1 kcat (s ) 270 480 830 160 44.1 34.6 259.8 114.8 ± ± ± ± 2.7 1.0 26.7 2.6 kcat/K mNADPH : kcat/K mNADH 0.0075 0.0097 0.0799 0.0627 760 763 31.3 35.1 assays were repeated using potassium phosphate (50 mM, pH 7.0) instead of Tris/HCl Results were very similar to those obtained in Tris/HCl for wild-type and mutant FAD domains, indicating that the presence of phosphate ions (that potentially could occupy the 2¢-phosphate binding site for the coenzyme) does not affect catalysis in nNOS FAD domain The relative catalytic efficiencies with NADPH and NADH as electron donors (kcat/Km[NADH]/kcat/Km[NADPH]) for the nNOS FAD domains are shown in the final column of Table (NADPH/NADH) and detail the extent of the ÔswitchÕ in specificity for the two pyridine nucleotide coenzymes These data reveal that there is negligible alteration in the relative selectivity between wild-type and the F1395W mutant These data give confidence that the F1395W mutant can be used as a wild-type mimic in mechanistic studies to observe changes in the environment of the aromatic stacking residue close to the FAD [this residue must move to facilitate docking of NAD(P)H in its catalytically relevant conformation, and for hydride transfer to occur] A similar approach exploiting changes in tryptophan fluorescence has been used to characterize conformational events in human CPR [26,27] Although F1395W is virtually identical to wild-type as regards comparative efficiencies with NADH/NADPH, the F1395A/S mutants show large changes in favour of NADH (Table 1) The relative efficiencies (kcat/Km[NADH]/kcat/ Km[NADPH]) are changed > 24-fold for the F1395A variant, and > 21.5-fold for the F1395S mutant Clearly, the replacement of F1395 with nonaromatic residues has a major effect on the ability of the nNOS FAD domain to discriminate against NADH, and this leads to large improvements in efficiency of F1395A/S mutants in catalytic turnover with NADH The steady-state kinetic data indicate that small overall changes in the Km values for NADH contribute partially to the specificity switch towards NADH, but that the major effect is on the kcat parameter (Table 1) A further interesting observation from these kinetic data is that the replacement of the aromatic stacking residue does not lead to enhanced binding of NADPH, contrary to observations made with the related pea ferredoxin reductase and human CPR enzymes [28,30] Clearly this reflects differences in structural features of the NAD(P)H binding site in NOS This is maybe not surprising in view of the known differences by which electron transfer is regulated in NOS isoforms compared Ó FEBS 2004 Kinetic and thermodynamic properties of nNOS FAD domains (Eur J Biochem 271) 2553 with other ferredoxin reductase and diflavin reductase enzymes Thermodynamic basis for electron transfer Anaerobic spectroelectrochemical methods were used to determine the reduction potentials for the FAD flavin in wild-type and all three F1395 mutants In all cases, the development of a spectral signal typical of a neutral, blue semiquinone was observed during the course of the redox titration, with absorption maximum close to 600 nm As in previous studies of the redox properties of human CPR, isosbestic points were observed in the titrations, at  500 nm (oxidized-to-semiquinone transition) and at  411 nm (semiquinone-to-hydroquinone transition) [36] Plots of the change in absorption vs reduction potential were made at both 475 nm and 600 nm in order to determine the midpoint reduction potentials for the flavin transitions Data are presented in Table The midpoint potentials for the oxidized/semiquinone (E1) and semiquinone/hydroquinone (E2) couples in the mutants are not grossly altered from wild-type values No hysteresis was observed in any of the titrations, and similar spectra were obtained at the same potentials in both reductive and oxidative directions The E1 values for the F1395S/A Table Reduction potentials for the FAD cofactor in wild-type nNOS FAD domain, the F1395A/S/W mutants and related diflavin reductase FAD domains Reduction potentials for the oxidized/semiquinone (E1), semiquinone/hydroquinone (E2) and oxidized/hydroquinone couples of the wild-type and mutant forms of nNOS reductase FAD domain are shown Experiments and data fitting were performed as described in Experimental Procedures E1 and E2 values were determined by fitting A600 (near semiquinone absorption maximum) data to a twoelectron Nernst function, as described [36,38] The E12 value was derived by fitting the A475 (near oxidized flavin absorption maximum) data to the Nernst equation SHE, Standard hydrogen electrode Reduction potential (vs SHE) A475 A600 FAD domain E1 nNOS wild-type nNOS F1395A nNOS F1395S nNOS F1395W Intact nNOSa Human CPR Human MSR Human NR1 P450 BM3 )177 )157 )156 )167 )250 )286 )222 )315 )264 E2 ± ± ± ± 4 ± ± ± ± 6b 5d )310 )289 )306 )300 )260 )371 )288 )365 )375 E12 ± ± ± ± ± ± ± ± 7b 5d )229 )227 )237 )222 – )329 )272 )340 )320 ± ± ± ± 5 ± ± ± ± 7b 8c 5d 4e a Values for the FAD in intact rat nNOS were determined by simulations at various wavelengths No statistical errors are reported on these data [44]; b E1, E2 and E12 for human CPR FAD domain were determined similarly at 583 nm and 474 nm [36]; c E12 value for human MSR FAD comes from A450 analysis for the intact reductase [33]; d E1 and E2 data for human NR1 are from data fitting at 585 nm, with the E12 value being the midpoint of these E1 and E2 values [35]; and that for the FAD domain of P450 BM3; e E12 value cited for the FAD domain of P450 BM3 is the midpoint of the E1 and E2 values determined at 600 nm mutants ()156 mV/)157 mV) are slightly more positive than those of wild-type and F1395W enzymes ()177 mV and )167 mV, respectively), but the overall changes in the thermodynamic properties of the flavins are rather minimal, as judged by the close proximity of the midpoint potentials for the two-electron couples determined from the A475 data (Table 2) Replacement of an aromatic residue for the aliphatic serine/alanine thus has relatively little effect on the potentials for the FAD cofactor, and does not destabilize the semiquinone to any significant degree However, clear differences in spectral properties are observed between the aromatic (wild-type and F1395W) and nonaromatic (F1395A/S) mutants As discussed above, there are significant differences in the spectra for the oxidized forms of the enzymes (Fig 2), probably resulting from differences in the environment of the FAD cofactor in the different proteins These differences might also be manifest in the semiquinone forms, as the relative intensities of the semiquinone signal in F1395A/S diminished compared with those observed for wild-type and F1395W (Fig 3) This might reflect a change in the absorption characteristics (i.e extinction coefficient) of the semiquinone species in the nonaromatic mutants However, small changes in the separations between the E1 and E2 couples could also give rise to changes in the amounts of semiquinone signal detected during redox titration This aspect is under further investigation The semiquinone formation constants (K values) were determined as described previously [42,43] These yielded values of 177.5 for wild-type and F1395W mutants, 170.8 for F1395A, and 344.2 for F1395S These values reinforce the assertion that the neutral blue semiquinone in wild-type and mutant enzymes is strongly stabilized The proteins all showed some tendency to aggregate at lower potentials, as has been observed with the FAD domain of P450 BM3 [38], and this caused some problems in obtaining good quality data for the F1395A/S mutants at potentials < )350 mV, due to the rather small changes in absorption in the semiquinone absorption coupled to the development of some turbidity in the solutions Notwithstanding these problems, near-identical values were obtained from duplicated redox titrations in all cases, and values derived from fitting at two different wavelengths produced consistent results Thus, despite qualitative differences in the absorption properties of the flavins in these mutants, there are relatively small changes in the reduction potentials Consequently, alterations in the kinetic properties of the wild-type and mutant FAD domains can not simply be explained in terms of large-scale changes in the potentiometric properties of their cofactors Stopped-flow kinetic analysis of electron transfer Reduction of the wild-type and mutant FAD domains was investigated by stopped-flow methods using both a photodiode array detector and single wavelength detection The spectral changes accompanying flavin reduction following rapid mixing with NADPH are shown for the wild-type and F1395S FAD domains in Fig The spectral changes for the wild-type enzyme revealed a rapid bleaching of flavin absorption in the early time domain (Fig 4A) consistent with flavin reduction These spectral changes were followed by the development of long wavelength signature over an 2554 A J Dunford et al (Eur J Biochem 271) Ó FEBS 2004 Fig Potentiometric analysis of wild-type and F1395 mutant nNOS reductase FAD domains (A) Spectral changes observed during the redox titration of wild-type FAD domain ( 45 lM) The most intense spectrum is that for the oxidized enzyme Reduction is associated with bleaching of the absorption in the region of the two major absorption bands of the flavin, while there is development, and then decay, of absorption at longer wavelength due to the formation of the semiquinone species, followed by its reduction to hydroquinone Arrows indicate the direction of absorption in these regions of the spectrum during reductive titration The inset shows a fit of the A600 (semiquinone) data to a two-electron Nernst function, as described previously [35,37,40] (B) A similar set of spectra and the relevant A600 vs reduction potential data fit from the titration of the F1395S mutant ( 65 lM) extended time domain, indicating the formation of a blue neutral semiquinone species (Fig 4B) Qualitatively similar data were obtained for the F1395W mutant FAD domain By contrast, reduction of the F1395S FAD domain (and also the F1395A FAD domain; data not shown) is also relatively rapid (Fig 4C), but over an extended time base the semiquinone signature is not developed (Fig 4D) Given the inferior signal-to-noise ratio of the photodiode array detector compared with data acquisition at single wavelengths using a photomultiplier, we also performed single wavelength studies at 600 nm for the wild-type and F1395S FAD domains This confirmed that relatively small absorption changes that might indicate formation of a blue neutral semiquinone are not observed at 600 nm for the F1395S (Fig 4C,D, insets) and F1395A (data not shown) domains Flavin reduction in the wild-type (Fig 4A, inset) and F1395W (data not shown) domains is accompanied by a rapid bleaching of absorption at 600 nm, which we attribute to the loss of charge-transfer character in an E–NADPH complex The fast formation and decay of a charge-transfer species at 600 nm in wild-type and F1395W FAD domains is consistent with our previous assignment of rapid 600 nm absorption changes in the isolated reductase domain of rat nNOS (which were likewise attributed to formation and decay of a charge-transfer species [20]) Reactions over an extended time base illustrate the formation of the blue semiquinone species (Fig 4B, inset) The lack of any spectral change in the early time domain for the F1395S and F1395A domains suggests that a spectrally distinct charge-transfer species is not formed (Fig 4C, inset) Observed reaction rates for flavin reduction were calculated by analysis of single wavelength reaction transients recorded at 454 nm (Fig 5) Reaction transients were biphasic, and data were analysed using a standard doubleexponential function; the amplitude of the first phase was found to contribute  70% of the total absorption change This phase was shown to represent hydride transfer from NADPH to FAD through stopped-flow fluorescence analysis, since the loss of NADPH fluorescence accompanying oxidation of the coenzyme on mixing wild-type domain with NADPH occurs with similar kinetics to the fast kinetic Ó FEBS 2004 Kinetic and thermodynamic properties of nNOS FAD domains (Eur J Biochem 271) 2555 Fig Spectral changes observed during the reduction of wild-type and F1395S FAD domain on mixing with NADPH Photodiode array data collected were obtained for the reaction of wild-type (10 lM) and F1395S (10 lM) FAD domain mixed with NADPH (100 lM) Reactions were performed in 50 mM Tris/HCl pH 7.4, 10% glycerol at 25 °C Upper panels: data for wild-type FAD domain recorded from 1.28 ms to s (A) and from 1.28 ms to 200 s (B) Lower panels: data for F1395S FAD domain recorded from 1.28 ms to s (C) and from 1.28 ms to 200 s (D) Insets show typical stopped-flow transients monitored at 600 nm for reactions of wild-type (5 lM, A and B) and F1395S (5 lM, C and D) FAD domains with NADPH (100 lM) Transients were recorded over the same time periods as the respective spectral changes shown in the main panels phase observed in absorption measurements Fig 6A The fluorescence transient also has a second phase with kinetics comparable to the slow phase seen in absorption mode, suggesting further oxidation of NADPH The mechanistic origin of the second phase is uncertain, but one possibility is that the slow phase might represent further reduction of the flavin following release of NADP+, as the equilibrium distribution of enzyme species adjusts to favour further reduction of the enzyme Similar arguments have been advanced in studies of the isolated FAD domain of human CPR [26] Alternatively, different conformational states of the FAD domain might also account for the biphasic nature of the reaction transients and this would be consistent with the observed NADH concentration dependence for the fast and slow phases in flavin reduction using NADH as reducing coenzyme (see below) Although the mechanistic origin of the two phases remains uncertain, it is clear that both phases report on flavin reduction by reducing coenzyme The observed rates of flavin reduction (fast phase) as a function of NADPH concentration are plotted in Fig 6B As with the isolated diflavin reductase of rat nNOS [20], these rates are independent of NADPH concentration in the pseudo first-order regime Kinetic constants for reactions with NADPH are collated in Table We also performed stopped-flow studies of flavin reduction in wild-type and mutant FAD domains using NADH as the reducing coenzyme Reaction transients were again biphasic (fast phase  70% and slow phase  30% of the total amplitude change) Unlike with NADPH, observed rates for each phase displayed a hyperbolic dependence on NADH concentration Derived kinetic constants for each phase of the reaction transient are collated in Table The most striking result from these series of stopped-flow studies is that the fast phase for the F1395A and F1395S FAD domains is faster than the wild-type and F1395W FAD domains (Table 3) Likewise, the slow phase of the kinetic transient is markedly more rapid in the F1395A and F1395S domains compared with the wild-type and F1395W Ó FEBS 2004 2556 A J Dunford et al (Eur J Biochem 271) Fig Stopped-flow transients of wild-type and mutant FAD domains monitored at 454 nm Kinetic transients show the reaction of each FAD domain (5 lM) with NADPH (100 lM) Reactions were performed in 50 mM Tris/HCl pH 7.4, 10% glycerol at 25 °C In all cases transients were fitted to a standard biphasic expression; fits are shown in A–D (A) Wild-type domain (B) F1395W (C) F1395A (D) F1395S domains, suggesting that the NMN ring of NADH is in a more favourable geometry for hydride transfer to the FAD following removal of the aromatic shielding residue With human CPR we have demonstrated that the FAD shielding residue (Trp676) is conformationally mobile using fluorescence stopped-flow methods [26,27] Fluorescence stopped-flow studies with the wild-type nNOS FAD domain indicated essentially no change in tryptophan emission on mixing with NADPH, consistent with the lack of a tryptophan residue in the NADPH-binding site Large changes in tryptophan fluorescence emission were observed, however, on mixing the F1395W FAD domain with NADPH Two kinetic phases were observed: the first (increase in fluorescence,  200 s)1) occurs on a timescale consistent with the kinetics of flavin reduction; the second phase (decrease in fluorescence emission, 0.04 s)1) accompanies development of the flavin semiquinone observed in stopped-flow absorption measurements (see Fig insets and Table 3) Clearly, the environment of Trp1395 in the mutant FAD domain is perturbed on reduction of the flavin, and also on subsequent disproportionation of the domain to yield the blue neutral semiquinone form Discussion This study on the kinetic and thermodynamic features of the wild-type and F1395 mutants of nNOS reductase indicates an important role for the aromatic residue that shields FAD in rat nNOS However, mutation of this residue in rat nNOS and the equivalent residue in other members of the diflavin reductase family has revealed different functional characteristics Previous studies on both human CPR and NOS revealed that the replacement of this aromatic residue with nonaromatic substitutes influenced the specificity for the reducing pyridine nucleotide coenzyme in favour of NADH [28,29] An  1000-fold switch in coenzyme specificity was achieved for the W676A mutant of human CPR [28] Also, in recent studies, we have demonstrated that a similar switch in specificity occurs in another member of the diflavin reductase family, flavocytochrome P450 BM3 (R Neeli, O Roitel, N S Scrutton and A W Munro, unpublished data) Herein, we show that the switch in specificity towards NADH occurs in the nNOS FAD domain, in which the aromatic shielding residue has been mutated to an aliphatic side chain, although the extent of Ó FEBS 2004 Kinetic and thermodynamic properties of nNOS FAD domains (Eur J Biochem 271) 2557 Fig Reduction of wild-type FAD domain monitored by absorption and fluorescence spectroscopy The reduction of wild-type FAD domain (5 lM) was monitored by absorption spectroscopy (454 nm) and fluorescence emission spectroscopy (excitation 340 nm) Reactions were performed in 50 mM Tris/HCl pH 7.4, 10% glycerol at 25 °C The reaction transient in absorption mode (A, trace a) is fitted to a two-exponential equation [ 450 s)1 ( 70% of total amplitude) and 95 s)1 ( 30% amplitude), respectively] Likewise, the reaction transient in fluorescence mode (A, trace b) is fitted to a two-exponential equation [ 400 s)1 ( 60% of total amplitude) and 90 s)1 ( 40% amplitude), respectively] (B) Plot of the observed rate constant kobs for the fast phase of flavin reduction as a function of NADPH concentration for wild-type and mutant FAD domains Conditions as for (A) The plot shows that, in all cases, the rate is independent of the concentration of NADPH used Symbols: (j) WT; (d) F1429W; (m) F1429A; (.) F1429S the conversion is less marked than that observed for the fulllength nNOS F1395A mutant in previous studies [29] An  25-fold switch in specificity towards NADH is achieved for the F1395A FAD domain [by comparing relative kcat/ Km values for NAD(P)H-dependent ferricyanide reduction] (Table 1) Our reductionist approach in studying the isolated FAD domain of nNOS has enabled us to analyse in detail the effects of these mutations in the absence of other influences (most notably those induced by CaM binding and by interflavin electron transfer) This has clear advantages over studies with the full-length NOS protein, which are compromised by multiple spectroscopic signals from additional cofactors and conformational effects induced by CaM binding Thus, for the first time we are able to report the effects of these mutations on the thermodynamic properties of the FAD and also their consequences on the kinetics of FAD reduction The thermodynamic properties of the wild-type and mutant nNOS FAD domains are compared with the potentials for the FAD domains of other members of the diflavin reductase class of enzymes in Table While there are clearly distinct differences in the absorption properties of the FAD on removal of the aromatic shielding residue (Fig 2), there are only relatively small differences in the thermodynamic properties of the wild-type and mutant FAD domains (a maximum of 21 mV between E1 couples, 21 mV between E2 couples, 15 mV between E12 couples) Potentiometric studies suggest that differences in spectral properties are also a feature of the semiquinone forms of the FAD, with the decreased intensity of the semiquinone observed in redox titrations of the F1395A/S mutants possibly being attributed to an approximately twofold change in the extinction coefficient for this species in these mutant domains When compared with the other diflavin reductases, there are very clear differences in the thermodynamic properties of the nNOS FAD flavin All of these proteins have in common the ability to stabilize the FAD blue semiquinone, but the wild-type and mutant nNOS domains have a more positive potential for the oxidized/ semiquinone couple (E1) compared with other members of the family Specifically, for wild-type nNOS FAD domain, the E1 value is between 45 and 138 mV more positive than E1 for the other flavoproteins (Table 2) By contrast, the potentials for the nNOS FAD domain semiquinone/ hydroquinone couples (E2) are very similar to those for the other diflavin reductases The net effect is that the overall potential for the oxidized/hydroquinone couple (E12) of wild-type nNOS FAD domain is 43–111 mV more positive than any of the other diflavin reductase enzymes A further point to note from the potentiometric analysis of the wild-type and mutant domains is that there is some variance with the previously reported data for the potentials of the wild-type nNOS FAD measured in the reductase (diflavin) domain of the enzyme [32] The data reported here for the FAD domain are E1 ¼ )177 ± mV; E2 ¼ )310 ± mV; (E12 ¼ )229 ± mV), whereas the values reported from an absorption vs potential fit to the inherently more complex four-electron Nernst function are E1 ¼ )232 ± mV; E2 ¼ )280 ± mV (E12 ¼ )256 ± mV) In recent studies, Gao et al have used even more complex simulations to derive estimates of the flavin and haem reduction potentials in intact nNOS While Ó FEBS 2004 2558 A J Dunford et al (Eur J Biochem 271) Table Kinetic constants and apparent enzyme–NADH dissociation constants obtained from analysis of stopped-flow kinetic data for wild-type and mutant FAD domains following mixing with NADPH and NADH For reactions with NADPH, kobs1 and kobs2 are independent of NADPH concentration over the range used in stopped-flow studies and represent rate constants for the fast and slow phases, respectively, observed at 454 nm; kobs3 is the observed rate constant for the formation of the neutral blue semiquinone For reactions with NADH, data for the fast phase and slow phase observed at 454 nm are shown klim is the limiting rate constant for flavin reduction and KNADH is the coenzyme concentration that yields klim/2 Values were derived from hyperbolic fits of rate data vs NADH concentration for wild-type and mutant FAD domains All reactions were performed in 50 mM Tris/HCl, 10% glycerol, pH 7.4, at 25 °C for wild-type and mutant enzymes, respectively, as indicated in Experimental procedures N/A, not applicable due to lack of semiquinone formation NADH NADPH Fast phase Slow phase kobs1 (s)1) WT F1395W F1395S F1395A kobs2 (s)1) kobs3 (s)1) klim1 (s)1) KNADH (mM) klim2 (s)1) KNADH (mM) 496 500 125 680 95 89 10 55 0.031 ± 0.002 0.032 ± 0.002 N/A N/A 82.6 70.3 114.0 139.6 6.2 25.7 3.5 9.3 3.8 5.6 67.5 91.0 1.40 2.9 7.8 29.9 ± ± ± ± 29 31 43 ± ± ± ± standard errors are not reported on these data, the results are broadly similar to those reported previously for the diflavin reductase form [32,44] It is possible that the further genetic dissection of the reductase to produce the isolated FAD domain results in some perturbation of the redox properties of the flavin However, no such changes were observed in similar domain dissection and potentiometric studies of the P450 BM3 diflavin reductase [38], although a small shift in potential was reported for the FAD domain in studies of full-length MSR and its genetically excised FAD domain [33] That said, regardless of the origin of the apparent small difference in the potentials of the FAD cofactor in full-length nNOS reductase and the isolated FAD domain, it is clear that the mutations not perturb the overall redox properties of the FAD (E12) to any significant degree The origin of the kinetic differences between wild-type and mutant FAD domains is therefore unlikely to be attributable to altered thermodynamic effects Exchange of the aromatic shielding residue (Trp676) in human CPR for alanine substantially compromises ( 400fold) the rate of FAD reduction by NADPH [27] This contrasts markedly with the properties of the F1395A and F1395S nNOS FAD domains, in which the rates of FAD reduction are similar to wild-type and F1395W FAD domain In stopped-flow studies, reduction of the isolated FAD of CPR is dominated by a slow kinetic phase ( 3.5 s)1), and it has been postulated that FAD reduction in this phase is linked to the release of NADP+ that displaces the equilibrium distribution of enzyme species towards further flavin reduction [26,45] A smaller but more rapid phase ( 200 s)1) that precedes the major flavin reduction step involves the establishment of an equilibrium distribution predominantly involving an enzyme–NADPH charge-transfer species and a small amount of reduced FAD domain bound to NADP+ In CPR, Trp676 is required to accelerate NADP+ release; in the W676H FAD domain of CPR, a reduced enzyme-NADP+ charge-transfer species is stabilized compared with wild-type FAD domain, indicating that Trp676 is actively involved in displacing NADP+ from reduced FAD domain [27] Mutagenesis of the aromatic shielding tyrosine residue in pea ferredoxinNADP+ reductase to serine stabilizes the E-NADP+ complex and allows the nicotinamide ring of the coenzyme ± ± ± ± 4.3 3.2 6.7 12.3 ± ± ± ± 0.8 1.7 0.6 1.3 ± ± ± ± 0.2 0.5 4.4 9.5 ± ± ± ± 0.4 0.7 1.2 6.7 to bind productively, close to the isoalloxazine ring [46] This does not occur in wild-type enzyme, indicating that the aromatic shielding residue competes with the nicotinamide ring for the productive binding site By analogy, one might expect F1395 in nNOS reductase to compete with nicotinamide coenzyme for productive binding, and that productive binding might be favoured in the F1395S and F1395A FAD domains In full-length nNOS reductase, however, the situation is more complicated owing to the effects of CaM on conformational events in the nicotinamide-binding site The recent studies of Adak et al indicate differential effects of CaM binding on full-length wild-type NOS and fulllength F1395S NOS, suggesting interplay between CaM binding, nicotinamide binding and F1395 in coenzyme binding/FAD reduction [29] The switch in coenzyme specificity towards NADH in CPR is consistent with a bipartite mode of binding nicotinamide coenzyme and weaker binding of NADH in wild-type enzyme compared with the W676A enzyme, owing to the flipping motion of W676 [27] By analogy with CPR, one might expect F1395 to flip on binding nicotinamide coenzyme In the F1395S and F1395A domains this model would suggest that the thermodynamically unfavourable flipping motion would no longer be required, and both binding sites in the bipartite recognition site for nicotinamide coenzyme should be readily accessible in the F1395S and F1395A domains In the wild-type and F1395W domains, interaction with the adenosine ribose moiety is probably the first binding step for the coenzyme, with subsequent occupation of the nicotinamide site occurring only after flipping of the aromatic shielding residue However, although the removal of the aromatic residue in the F1395S and F1395A FAD domains clearly switches the coenzyme specificity towards NADH, the effect is predominantly attributed to an improved turnover number with NADH and not an improved Michaelis constant, unlike with CPR [28] This suggests that a more favourable binding geometry for hydride transfer to FAD, and not an improved affinity for NADH, is the key determinant in switching specificity This assertion is consistent with the improved rate constants observed in stopped-flow studies for reduction of the F1395S and F1395A FAD domains by NADH (Table 3) Also, the Michaelis constant for NADPH is not Ó FEBS 2004 Kinetic and thermodynamic properties of nNOS FAD domains (Eur J Biochem 271) 2559 substantially affected on removal of the aromatic shielding residue (Table 1), and this is in stark contrast with the effects on Km on mutating Trp676 to alanine in human CPR with both NADPH and NADH which resulted in  75-fold and 150-fold decrease in Km, respectively Again, this emphasizes in nNOS FAD domain that the geometry for hydride transfer from NADH to FAD is improved on removing the aromatic shielding residue, and that differential binding affinities are not the major factor in coenzyme discrimination In conclusion, using a domain dissection approach we have been able to demonstrate the functional consequences of removal of the conserved aromatic shielding residue in nNOS FAD domain Our approach has released us from complications associated with CaM binding and internal electron transfer to the FMN and haem domains In contrast with work on CPR, removal of the shielding residue does not adversely affect the kinetics of flavin reduction, although mutation results in a switch in coenzyme specificity as reported also for human CPR The basis of this switch appears to have its origins in an improved geometry for NADH binding rather than on improved affinity for the coenzyme (as seen with CPR) Mutagenesis to a 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F1395A) and by probing the thermodynamic and kinetic consequences of these mutations We have studied the thermodynamic and kinetic. .. determination of the midpoint reduction potentials of flavin cofactors, and also of the haems in the case of NOS and P450 BM3 In spectroelectrochemical titrations of the isolated domains, the lack of. .. Fig The coenzyme-binding site in nNOS FAD domain and sequence alignments around the conserved aromatic residue in this domain (A) The nicotinamide-binding site of nNOS FAD domain showing the