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 exch
Trang 1Thermodynamic 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
reduc-tase and novel reducreduc-tase 1), and tyrosine in plant
ferredoxin-NADP+ 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
wild-type Stopped-flow studies indicate flavin reduction by
NADH is significantly faster with F1395S and F1395A
domains, suggesting that this contributes to altered
prefer-ence in coenzyme specificity Unlike cytochrome P450
reductase, the switch in coenzyme specificity is not attributed
to differential binding of NADPH and NADH, but prob-ably results from improved geometry for hydride transfer in the F1395S– and F1395A–NADH complexes Potentio-metry indicates that the substitutions do not significantly perturbthermodynamic 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 incuba-tion 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
Keywords: coenzyme specificity; cytochrome P450 reduc-tase; electron transfer; nitric oxide synthase; redox potential
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
cancer-associated 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)-5,6,7,8-tetrahydrobiopterin [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
bind-ing has been proposed to enhance the rate of interflavin
electron transfer [17–19], although this remains a contro-versial 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-depend-ent 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 reduc-tion 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)
Trang 2release 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
conjec-tured to stabilize interaction with the nicotinamide group
common to both NADH and NADPH [30]
In rat nNOS, residue Phe1395 is located close to the
reface 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
mamma-lian 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
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 reduc-tases 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 assign-ment 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 trigger mechanism that positively or
Fig 1 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
equiv-alent W677 in rat CPR The FAD of CPR
(PDB code 1AMO) and rat NOS (PDB code
1F2O) are shown in yellow NADP + in the
off conformation (PDB code 1JA1) is shown
in blue, and in the on 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
di-flavin reductases and plant ferredoxin
reduc-tase in the region of the conserved aromatic
residues that shield the FAD isoalloxazine
ring nNOS, rat neuronal nitric oxide
thase; eNOS, rat endothelial nitric oxide
syn-thase; iNOS, rat inducible nitric oxide
synthase; CPR, human cytochrome P450
re-ductase; 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.
Trang 3negatively 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 sub tle 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,
for-ward 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¢
toge-ther 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 95C for 30 s followed b y
16 cycles of 95C for 30 s, 55 C for 1 min and 68 C for
9 min Nonmutated template DNA was then removed by DpnI digestion and mutant DNA transformed into Escheri-chia 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 30C Harvested cells were resuspended in lysis b uffer [50 mL; 50 mM Tris/HCl pH 7.4 containing 10% (v/v) glycerol, 1 mMCaCl2and a CompleteTM EDTA-free 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 (0–0.5M) of KCl Fractions containing FAD domain were pooled, and applied to an affinity resin (2¢5¢-ADP Seph-arose) equilibrated with lysis buffer containing 100 mM NaCl After washing ( 250 mL lysis buffer 100 mMNaCl 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 exhaust-ively 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 < 2 p.p.m The protein solution [ 50 lMin
5 mL 100 mMpotassium phosphate pH 7.0 in the presence and absence of 10% (v/v) glycerol] was titrated electro-chemically according to the method of Dutton [41] using sodium dithionite as reductant and potassium ferricyanide
as oxidant Mediators (2 lMphenazine methosulfate, 5 lM 2-hydroxy-1, 4-naphthoquinone, 0.5 lM methyl viologen, and 1 lMbenzyl viologen) were included to mediate in the range between +100 and)480 mV as described previously [35,41] At least 15 min 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 electro-chemical potential of the solution was measured using a Hanna pH 211 meter coupled to a Pt/Calomel electrode (Thermo Russell Ltd) at 25C The electrode was calibrated
Trang 4using 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 intoORIGIN(Microcal) and spectral
subtrac-tions 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
Technol-ogy) Measurements were carried out at 25C in 50 mM
Tris/HCl pH 7.4 containing 10% (v/v) glycerol Protein
concentration was 5 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 mMTris/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
(trypto-phan) Emission bands were selected using the appropriate
band pass filter
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
spectro-photometer Assays were performed in the double-beam
spectrophotometer to take account of any
nonenzyme-mediated reduction of electron acceptor The reference
cuvette contained the same mix as the sample cuvette, but
the same volume of b uffer 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)¼ 1020M )1Æcm)1) Reactions were
performed in 50 mM Tris/HCl pH 7.4, 10% glycerol at
25C 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
concen-tration of ferricyanide (2 mM).ORIGINsoftware (Microcal)
was used in data fitting to derive Kmand kcatvalues from
steady-state assays
Results Domain isolation and mutagenesis of Phe1395 Each of the F1395S/A/W FAD domains were purified in high yield (typically 15 mg from 1 L recombinant cells) and
in pure form as judged by SDS/PAGE The spectral properties of each are shown in Fig 2 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 inten-sified 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 conse-quence of the nonaromatic amino acid substitutions in F1395A/S are consistent with a less hydrophobic environ-ment 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 Kmfor NADPH induced by the replacement of
Fig 2 UV-Visible absorption spectra for wild-type and mutant forms of nNOS FAD domain (all 7 l M ) Protein samples were contained in
50 m M 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).
Trang 5the FAD-stacking phenylalanine with either aromatic
(tryptophan) or nonaromatic (alanine, serine) sidechains
(Table 1) There is an apparent small decrease in KMfor
NADPH in the F1395W mutant (Km¼ 15.4 lMcf 28.2 lM
for wild-type) and a larger increase for the F1395A mutant
(Km¼ 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 kcatfor the F1395W and F1395S mutants compared to
wild-type (70% and 31%, respectively, of wild-type kcat);
interestingly, the kcatfor F1395A is increased by 30% over
wild-type The net effects on the second order rate constant
(kcat/Km) describing the overall efficiency of
NADPH-dependent ferricyanide reduction is that the F1395W
mutant shows a modest improvement (30%) over
wild-type, 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 Kmvalues are lower in all mutants
than in wild-type, with F1395S showing the greatest
improvement (Km¼ 1830 lMcompared with 5890 lMfor
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/Kmratio for the F1395A/S
mutants over wild-type (10.7/8.4-fold), with a minor
improvement also observed for the F1395W mutant
(1.3-fold) (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 kcatfor NADH-dependent ferricyanide reduction
The rather smaller enhancement of kcatreported 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
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 1 (NADPH/NADH) and detail the extent of the
switch 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 cata-lytic 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 kcatparameter (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
Table 1 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 K m and k cat values for ferricyanide (Fe[(CN) 6 ] 3– ) were determined at a fixed and saturating concentration of NADPH (500 l M ) The K m and k cat values for NADPH and NADH were determined at a fixed and saturating concentration of ferricyanide (2 m M ) All reactions were performed in 50 m M Tris/HCl, 10% glycerol, pH 7.4, at 25 C.
nNOS
K m (l M ) k cat (s)1)
k cat /K m
(l M )1 Æs)1) K m (l M ) k cat (s)1)
k cat /K m
(l M )1 Æs)1)
k cat /K NADPH
k cat /K NADH m
Trang 6with 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 2 The midpoint
potentials for the oxidized/semiquinone (E1) and
semiqui-none/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
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 A475data (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 signi-ficant 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 deter-mined 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 dupli-cated redox titrations in all cases, and values derived from fitting at two different wavelengths produced consistent results Thus, despite qualitative differences in the absorp-tion properties of the flavins in these mutants, there are relatively small changes in the reduction potentials Conse-quently, 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 photo-diode 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 4 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
Table 2 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 (E 1 ),
semiquinone/hydroquinone (E 2 ) 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 E 1 and E 2 values were determined by
fitting A 600 (near semiquinone absorption maximum) data to a
two-electron Nernst function, as described [36,38] The E 12 value was
de-rived by fitting the A 475 (near oxidized flavin absorption maximum)
data to the Nernst equation SHE, Standard hydrogen electrode.
FAD domain
Reduction potential (vs SHE)
)365 ± 5 d
)340 ± 5 d
a
Values for the FAD in intact rat nNOS were determined by
si-mulations at various wavelengths No statistical errors are reported
on these data [44]; b E 1 , E 2 and E 12 for human CPR FAD domain
were determined similarly at 583 nm and 474 nm [36];cE 12 value
for human MSR FAD comes from A 450 analysis for the intact
reductase [33]; d E 1 and E 2 data for human NR1 are from data
fitting at 585 nm, with the E 12 value being the midpoint of these E 1
and E 2 values [35]; and that for the FAD domain of P450 BM3;
e E 12 value cited for the FAD domain of P450 BM3 is the midpoint
of the E and E values determined at 600 nm.
Trang 7extended 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 forma-tion 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 calcu-lated by analysis of single wavelength reaction transients recorded at 454 nm (Fig 5) Reaction transients were biphasic, and data were analysed using a standard double-exponential 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 ana-lysis, 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
Fig 3 Potentiometric analysis of wild-type and F1395 mutant nNOS reductase FAD do-mains (A) Spectral changes observed during the redox titration of wild-type FAD domain ( 45 l M ) The most intense spectrum is that for the oxidized enzyme Reduction is associ-ated 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 wave-length due to the formation of the semiqui-none 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 A 600 (semiquinone) data to a two-elec-tron Nernst function, as described previously [35,37,40] (B) A similar set of spectra and the relevant A 600 vs reduction potential data fit from the titration of the F1395S mutant ( 65 l M ).
Trang 8phase 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
coen-zyme 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 3
We also performed stopped-flow studies of flavin reduc-tion 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 3 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
Fig 4 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 l M ) and F1395S (10 l M ) FAD domain mixed with NADPH (100 l M ) Reactions were performed in 50 m M Tris/HCl pH 7.4, 10% glycerol at 25 C Upper panels: data for wild-type FAD domain recorded from 1.28 ms to 1 s (A) and from 1.28 ms to 200 s (B) Lower panels: data for F1395S FAD domain recorded from 1.28 ms to 1 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 l M , A and B) and F1395S (5 l M , C and D) FAD domains with NADPH (100 l M ) Transients were recorded over the same time periods as the respective spectral changes shown in the main panels.
Trang 9domains, 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)
accom-panies development of the flavin semiquinone observed in
stopped-flow absorption measurements (see Fig 4 insets
and Table 3) Clearly, the environment of Trp1395 in the
mutant FAD domain is perturbed on reduction of the
fla-vin, 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 speci-ficity 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
Fig 5 Stopped-flow transients of wild-type and mutant FAD domains monitored at 454 nm Kinetic transients show the reaction of each FAD domain (5 l M ) with NADPH (100 l M ) Reactions were performed in 50 m M 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.
Trang 10the conversion is less marked than that observed for the full-length 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/
Kmvalues 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 2 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 E1couples,
21 mV between E2 couples, 15 mV between E12couples) 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 thermo-dynamic 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 E1value 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 E1¼)177 ± 5 mV; E2¼ )310 ± 8 mV; (E12¼)229 ± 5 mV), whereas the values reported from an absorption vs potential fit to the inherently more complex four-electron Nernst function are E1¼)232 ± 7 mV; E2¼)280 ± 6 mV (E12¼ )256 ± 7 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
Fig 6 Reduction of wild-type FAD domain monitored by absorption
and fluorescence spectroscopy The reduction of wild-type FAD
domain (5 l M ) was monitored by absorption spectroscopy (454 nm)
and fluorescence emission spectroscopy (excitation 340 nm) Reactions
were performed in 50 m M 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
tran-sient 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 k obs for
the fast phase of flavin reduction as a function of NADPH
concen-tration 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.