KasperAnna L. Shen, Daniel S. Sem and Charles B.  Oxidoreductase

P450Half-reaction of NADPH-Cytochrome Mechanistic Studies on the ReductiveENZYMOLOGY:

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Mechanistic Studies on the Reductive Half-reaction ofNADPH-Cytochrome P450 Oxidoreductase*

(Received for publication, September 21, 1998, and in revised form, November 27, 1998)

Anna L. Shen, Daniel S. Sem, and Charles B. Kasper‡

From the McArdle Laboratory for Cancer Research, Madison, Wisconsin 53706

Site-directed mutagenesis has been employed to studythe mechanism of hydride transfer from NADPH toNADPH-cytochrome P450 oxidoreductase. Specifically,Ser457, Asp675, and Cys630 have been selected because oftheir proximity to the isoalloxazine ring of FAD. Substi-tution of Asp675 with asparagine or valine decreasedcytochrome c reductase activities 17- and 677-fold, re-spectively, while the C630A substitution decreased en-zymatic activity 49-fold. Earlier studies had shown thatthe S457A mutation decreased cytochrome c reductaseactivity 90-fold and also lowered the redox potential ofthe FAD semiquinone (Shen, A., and Kasper, C. B. (1996)Biochemistry 35, 9451–9459). The S457A/D675N andS457A/D675N/C630A mutants produced roughly multi-plicative decreases in cytochrome c reductase activity(774- and 22000-fold, respectively) with correspondingdecreases in the rates of flavin reduction. For each mu-tation, increases were observed in the magnitudes of theprimary deuterium isotope effects with NADPD, consist-ent with decreased rates of hydride transfer fromNADPH to FAD and an increase in the relative ratelimitation of hydride transfer. Asp675 substitutions low-ered the redox potential of the FAD semiquinone. Inaddition, the C630A substitution shifted the pKa of anionizable group previously identified as necessary forcatalysis (Sem, D. S., and Kasper, C. B. (1993) Biochem-istry 32, 11539–11547) from 6.9 to 7.8. These results areconsistent with a model in which Ser457, Asp675, andCys630 stabilize the transition state for hydride transfer.Ser457 and Asp675 interact to stabilize both the transitionstate and the FAD semiquinone, while Cys630 interactswith the nicotinamide ring and the fully reduced FAD,functioning as a proton donor/acceptor to FAD.

The microsomal and nuclear envelope flavoprotein NADPH-cytochrome P450 oxidoreductase (P450R1; NADPH-ferrihemo-

protein oxidoreductase, EC 1.6.2.4; hereafter referred to asreductase), one of a family of FMN- and FAD-containing en-zymes that includes nitric-oxide synthase, the sulfite reductasea-subunit, and P450BM-3 (for recent reviews, see Refs. 1 and2), mediates the transfer of electrons from NADPH to cyto-chrome P450 and other microsomal proteins as well as to non-physiological electron acceptors such as cytochrome c and fer-ricyanide (3). Electron transfer to electron acceptors such ascytochromes c or P450 proceeds from NADPH to FAD to FMN,while ferricyanide and 3-acetylpyridine adenine dinucleotidephosphate (AcPyrADP) accept electrons directly from FAD (4–6).

Sequence comparisons as well as a variety of biochemicalstudies (6–11) have suggested the presence of independentFMN- and FAD-binding domains. The amino-terminal FMN-binding domain, with homology to the bacterial flavodoxins (7),is separated by an insertion sequence having no homology toany known protein from the FAD/NADPH binding domain,which is related to another class of flavoproteins, the transhy-drogenases (1, 2, 12), and includes ferredoxin-NADP1 reduc-tase (FNR), NADH-nitrate reductase, and NADH-cytochromeb5 reductase as well as phthalate dioxygenase reductase (13).Both FNR and P450R abstract the pro-R (A-side) hydrogen ofNADPH (12, 14), with the conformation of the bound nicotina-mide anti in P450R (14) but unknown for other members of thisfamily. These sequence similarities have been confirmed by therecently described x-ray crystal structure of P450R, which alsohighlights the role of the insertion sequence in aligning theflavin isoalloxazine rings in a position for direct electron trans-fer (15). Finally, potential flavin- and NADPH-binding, as wellas catalytic, residues identified by sequence comparisons ofP450R with flavodoxin and FNR (2, 7) have been confirmed bybiochemical studies, including site-directed mutagenesis andkinetic measurements (16–18), as well as crystallographicstudies (15).

The crystal structures of FNR and P450R place three con-served residues, Ser457, Cys630, and Asp675, in close proximityto the isoalloxazine ring of FAD. Ser457 is in a position to forma hydrogen bond with the oxidized or reduced flavin N-5 ofP450R, while the homologous Ser96 of FNR interacts with thereduced, but not the oxidized, flavin N-5, suggesting a role forthese residues in hydride transfer and/or stabilization of thereduced flavin (12, 15, 19, 20). We have previously shown thatsubstitution of Ser457 produces large decreases in the rate ofhydride transfer from NADPH to FAD and lowers the FAD/FADH zredox potential (18). pH studies have indicated thatcatalysis is dependent upon deprotonation of an acidic grouphaving a pKa between 6.2 and 7.3 (21); comparison with FNRsuggests that Asp675 could fill this role (2, 12). The currentstudy explores the roles of Cys630 and Asp675 of NADPH-cyto-chrome P450 oxidoreductase in catalysis and FAD reductionand shows that the three residues Ser457, Asp675, and Cys630

interact to form the catalytic site for hydride transfer fromNADPH to FAD.

* This research was supported by National Institutes of Health (NIH)Grants CA22484 and CA0920. NMR studies were carried out at theNational Magnetic Resonance Facility at Madison (operation subsi-dized by the NIH Biomedical Research Technology Program under NIHGrant RR02301; equipment funded by the University of Wisconsin,National Science Foundation (NSF) Academic Infrastructure Programunder NSF Grant BIR-9214394, the NIH Shared Instrumentation Pro-gram under NIH Grants RR02781 and RR08438, the NIH BiomedicalResearch Technology Program under NIH Grant RR02301, the NSFBiological Instrumentation Program under NSF Grant DMB-8415048,and the U.S. Department of Agriculture). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed.1 The abbreviations used are: P450R, NADPH-cytochrome P450 oxi-

doreductase; FNR, ferredoxin-NADP1 reductase; PCR, polymerasechain reaction; AcPyrADP, 3-acetylpyridine adenine dinucleotide phos-phate; e2, electron equivalent(s); CAPS, 3-(cyclohexylamino)propane-sulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 9, Issue of February 26, pp. 5391–5398, 1999© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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MATERIALS AND METHODS

Expression and purification of recombinant NADPH-cytochromeP450 oxidoreductase was carried out as described previously (16), ex-cept that cultures were grown and induced at 28 instead of 37 °C.Protein was assayed by the BCA method (22), and FMN and FAD weredetermined by the method of Faeder and Siegel (23). Cytochrome c,ferricyanide, and AcPyrADP activities were assayed as described pre-viously (24), except that reactions were initiated by the addition ofprotein. Steady-state kinetic parameters were determined in the pres-ence of 0.27 M potassium phosphate, pH 7.7.

The D675N and C630A mutants were constructed by the methodof Kunkel (25), using the following 59-oligonucleotides: D675N, ACT-CACTAAACGTGTGGAGC; C630A, TCTATGTGGCCGGGGATGC. TheD675V mutant was prepared by PCR (26), using the following oligonu-cleotides: D675V, GGGTCCTAGGTCCTAGCTCCACACAACTAGTGA-GTA; 1858, GTGTGAGCTGCTGCCACGCC. The D675V oligonucleotidecontains the indicated mutation and an AvrII site for cloning of themutant fragment. PCR reactions contained 10 mM Tris, pH 8.3, 50 mM

KCl, 6.5 mM MgCl2, 0.001% gelatin, 200 mM of each dNTP, 10 mg/mltemplate DNA, 1 mM D675V mutagenic oligonucleotide, 1 mM 1858oligonucleotide (hybridizing to bases 59 of the mutation site), and 0.05units AmpliTaq polymerase (Perkin-Elmer, Foster City, CA). Reactionconditions were as follows: 94 °C, 1 min; 47 °C, 1 min; 72 °C, 2 min; 25cycles, followed by extension for 5 min at 72 °C. Ampliwax (Perkin-Elmer) was used according to the manufacturer’s instructions. Afterremoval of free oligonucleotides by centrifugation through an Mr

100,000 cut-off filter (Millipore Corp., Bedford, MA), PCR-amplifiedDNA fragments were digested with AvrII and NheI, purified by agarosegel electrophoresis, and cloned into pOR263. All mutant plasmids werecharacterized by restriction mapping and sequencing of the PCR-am-plified regions and cloning sites. Sequencing was performed with theAmpliTaq cycle sequencing kit (Perkin-Elmer). Multiply substitutedmutants were constructed by restriction enzyme digestion and cloningof the mutant fragments into pOR263 and confirmed by sequencing.

For studies of the primary deuterium isotope effect, A-side NADPDwas synthesized and purified by high performance liquid chromatogra-phy as described previously (18). The fraction of deuteration was deter-mined as described previously (27) and ranged from 0.8 to 0.9. Cyto-chrome c assays for isotope effect studies were carried out in 10 mM

potassium phosphate, 0.45 M KCl, pH 7.7 (I1/2 5 478 mM). Primarydeuterium isotope effects were determined by the method of directcomparison (28) and fitted to the following equation, which assumesdifferent isotope effects on Vmax and Vmax/Km

NADPH (27, 28),

v 5V*A

Km*~1 1 Fi*EV/K! 1 A*~1 1 Fi*Ev!(Eq. 1)

where v is the initial velocity, A is the concentration of NADPH, Fi is thefraction of deuterium label, EV/K is the isotope effect minus 1 on Vmax/Km

NADPH, and Ev is the isotope effect minus 1 on Vmax.pH studies were carried out at an ionic strength of 475 mM. Buffers

were prepared as described previously (21). Assays at pH 10, 10.5, and11, were carried out in CAPS buffer. Data were fitted to Equations 2–5(30) as follows,

Y 5Y0

1 1 ~H/K1! 1 ~K2/H!(Eq. 2)

where Y is kcat or kcat/KmNADPH; H is the proton concentration; K1 and K2

are the acid dissociation constants for the acidic and basic groups,respectively; and Y0 is the value of Y when both groups are in theirpreferred ionization state,

Y 5YH

1 1 ~H/K1! 1 ~K2/H!1

YL

~K1/H! 1 1(Eq. 3)

Y 5~YL*~H/K1!! 1 YH

~H/K1! 1 1(Eq. 4)

where Y is kcat or kcat/KmNADPH; H is the proton concentration; K1 and K2

are the acid dissociation constants for the acidic and basic groups,respectively; and YH and YL are the values of Y at high and low pH,respectively, and

Y 5YM

1 1 ~H/K1! 1 ~K2/H!1

YH

1 1 ~H/K3! 1 ~K2/H!(Eq. 5)

where Y is kcat/KmNADPH, with a value of YM at intermediate pH and YH

at high pH, K1 and K3 are acid dissociation constants for the acidicgroups, and K2 is the acid dissociation constant for the basic group.

Equation 2 describes a model where one acidic group must be unpro-tonated and one basic group must be protonated for maximum activity.Equation 3 describes a model similar to Equation 2 but which retains alower level of activity upon protonation of the acidic group. Equation 4describes a model in which a single group must be unprotonated formaximum activity. Equation 5 describes a model where two acidicgroups, with different pKa values, must be unprotonated and one basicgroup must be protonated for maximum activity.

Measurements of visible absorption spectra and anaerobic NADPHreduction rates and anaerobic NADPH titrations were carried out at28 °C on a Beckman 7500 diode array spectrophotometer. Anaerobicreactions contained 50 mM Tris, pH 7.7, 10 mM glucose, 5u/ml glucoseoxidase, 1 mM methyl viologen, and P450R at the concentrations indi-cated (31). Cuvettes were sealed, and oxygen was removed by subjectingthe samples to several cycles of evacuation followed by flushing withargon purified by passage through a scrubbing tower containing 0.5%dithionite, 0.1% 2-anthroquinone sulfonate, and 0.4% NaOH. Aerobicstopped-flow studies were carried out on an Olis RSM 100 rapid scan-ning spectrophotometer. Reactions were carried out in 50 mM Tris, pH7.7, 28 °C, and data were fitted to the following equations,

At 5 A0*e~2k1*t! (Eq. 6)

At 5 ~A0*e~2k1*t!! 1 ~A1*e~2k2*t!! (Eq. 7)

where At is the absorbance at time t, and A0 and A1 are the initialabsorbance values for the first and second phases of reduction,respectively.

RESULTS

Spectral Properties—Fig. 1 shows the visible absorptionspectra of the oxidized wild-type and mutant reductases, withbroad peaks at approximately 380 and 452 nm similar to thatdescribed for the rat liver enzyme (4). The addition of NADPHunder aerobic conditions to each of the proteins produced spec-tra characteristic of the air-stable semiquinone (FAD/FMNHz),with a decreased absorbance at 452 nm and a long wavelengthabsorbance band having a maximum at approximately 585 nmand a shoulder at 630 nm. None of the substitutions producedsignificant changes in the visible absorption spectra. This is inagreement with results of flavin analyses, which indicated noeffect of any of the substitutions on FMN or FAD content.

Enzymatic Activity—Substitution of any one of the threeresidues Ser457, Asp675, or Cys630 produced large decreases inrates of reduction of cytochrome c, which accepts electrons fromFMN, and ferricyanide and AcPyrADP, each of which acceptselectrons from FAD, suggesting that these mutations interruptelectron transfer from NADPH to FAD. Specific activities in thepresence of 0.27 M potassium phosphate, pH 7.7, and saturat-ing substrate concentrations are shown in Table I. The largestdecrease in cytochrome c reductase activity was produced bythe D675V substitution, which had only 0.1% of wild-type ac-tivity. Activities with the substrates ferricyanide andAcPyrADP were decreased 85- and 39-fold, respectively. TheD675N substitution, which retains hydrogen-bonding capacity,produced smaller effects, with only a 14-fold decrease in cyto-chrome c reductase activity and 15- and 5-fold decreases, re-spectively, in activities with the substrates ferricyanide andAcPyrADP. Substitution of Cys630 with alanine produced a37-fold decrease in cytochrome c reductase activity and 30- and13-fold decreases, respectively, in electron transfer to ferricya-nide and AcPyrADP.

We have previously reported a 90-fold decrease in cyto-chrome c reductase activity as a result of the S457A substitu-tion (18). Combination of the S457A mutation with substitu-tions at Asp675 and Cys630 produced multiplicative effects oncytochrome c reductase activities. Cytochrome c reductase ac-tivity of the S457A/D675N double mutant was decreased ;600-fold, while cytochrome c reductase activity of the S457A/

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D675N/C630A triple mutant was very close to backgroundlevels, with a calculated specific activity more than 4 orders ofmagnitude less than wild type. Ferricyanide reductase activityof the S457A/D675N mutant was decreased to 6% of wild typeand AcPyrADP activity to 1% of wild type.

Steady-state Kinetics—Table II shows steady-state kineticparameters for the active-site mutants, with cytochrome c aselectron acceptor. As was demonstrated previously for theSer457 mutations (18), substitutions of Asp675 and Cys630 pro-duced large decreases in kcat, with no significant changes inKm

NADPH, indicating that these mutations affect catalysis onlyand not cofactor binding. As with the wild-type and S457Aenzymes, NADP1 was a competitive inhibitor versus NADPH;Ki

NADP1 was only minimally affected by substitutions of Asp675

and Cys630. Kmcyt c values decreased in parallel with the de-

creases in kcat, consistent with the nonclassical two-site ping-pong mechanism of P450R (32) and a decrease in the rate of thereductive half-reaction (33).

To assess the relative rate limitation of hydride transfer forthe active-site mutants, the deuterium isotope effect on cyto-chrome c reduction with [(4S-H),(4R-D)]NADPD was deter-mined. In agreement with previously reported results (18, 32),the wild-type deuterium isotope effect on Vmax was 2.7 at high

(478 mM) ionic strength, with a similar isotope effect observedon Vmax/Km

NADPH, consistent with hydride transfer being par-tially rate-limiting. Substitution of Asp675 increased the isotopeeffects on Vmax and Vmax/Km

NADPH to values ranging from 4 to7 (Table II). Although the activity of the C630A mutant wasabout 10% of wild type, the deuterium isotope effects on bothVmax and Vmax/Km

NADPH were increased markedly, to approx-imately 10. The double substitution, D675N/S457A, producedisotope effects similar to those of the S457A single mutant.

pH Dependence of Cytochrome c Reductase Activity—The pHdependence of wild-type kcat and kcat/Km

NADPH has been previ-ously shown to be dependent upon two ionizable groups. Thefirst has a pKa of 6.2 at 850 mM ionic strength and 7.1 at 350mM ionic strength, which must be unprotonated for maximumactivity, while the other has a pKa of approximately 8.8, whichmust be protonated for maximum activity (21). To determine ifthese pKa values could be attributed to one or more of theseactive-site residues, the pH dependence of kcat and kcat/Km

NADPH with cytochrome c as the substrate was determined.Fig. 2 shows the pH dependence of kcat and kcat/Km

NADPH forthe D675N, D675V, S457A/D675N, and C630A mutants. ThepH dependences of wild-type kcat and S457A cytochrome creductase activity are also presented. At 475 mM ionic strength,

FIG. 1. Visible absorption spectra ofwild-type and mutant P450R pro-teins. Semiquinone spectra were ob-tained by adding NADPH under aerobicconditions and recording after equilib-rium had been reached (.15 min). A, wildtype, 32 mM; B, D675V, 18 mM; C, D675N,13 mM; D, C630A, 20 mM; E, S457A/D675N, 11 mM; F, S457A/D675N/C630A,22 mM. Solid lines, oxidized; broken lines,semiquinone.

TABLE ISpecific activities of active-site mutants

ProteinSpecific activitya

Cytochrome c Ferricyanide AcPyrADP

mmol/min/mg protein

Wild type 51.5 6 9.0 (13) 102 6 7.9 (4) 1.95 6 0.2 (3)D675N 3.8 6 0.05 (3) 6.8 6 1.5 (3) 0.43 6 0.02 (3)D675V 0.07 6 0.02 (3) 1.2 6 0.2 (3) 0.05 6 0.005 (3)C630A 1.4 6 0.4 (3) 3.4 6 0.3 (3) 0.15 6 0.04 (3)S457Ab 0.62 6 0.05 (7) 0.74 6 0.13 (4) 0.13 6 0.02 (3)S457A/D675N 0.09 6 0.01 (3) 1.6 (2) 0.02 6 0.002 (3)S457A/D675N/C630A 0.003 6 0.0009 (3) NDc ND

a Reactions were carried out in 0.27 M potassium phosphate, pH 7.7, 28 °C. Cytochrome c assays contained 50 mM NADPH and 65 mM cytochromec. Ferricyanide assays contained 100 mM NADPH and 500 mM potassium ferricyanide. Assays for reduction of AcPyrADP contained 100 mM NADPHand 200 mM AcPyrADP. Values are expressed as mean 6 S.D. (number of preparations analyzed).

b Taken from Ref. 18.c ND, not determined.

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wild-type kcat displayed pKa values of 6.9 6 0.1 and 9.6 6 0.1(Fig. 2A), consistent with previous results (29). The S457Amutant (Fig. 2B) displayed the same acidic pKa (6.8), but thepKa of the basic group was not seen over the range tested.Notably, the acidic pKa of 6.9 was not eliminated by the Asp675

substitutions, suggesting that some group other than Asp675 isresponsible for this pKa. The pH dependence of kcat and kcat/Km

NADPH for the D675N mutant (Fig. 2, C and D) was similarto that of wild type in the acidic limb, with acidic pKa values of7.1 6 0.2 and 6.8 6 0.3, respectively, but the basic pKa valueswere again shifted to .10. D675V kcat showed only a 2-foldchange over the pH range assayed; however, curve fitting in-dicated the presence of an acidic pKa of 6.6 6 0.2 and anincrease in the basic pKa (Fig. 2E). The pH dependence of

D675V kcat/KmNADPH also displayed the presence of an acidic

and a basic group, with pKa values of 6.0 6 0.2 and 10.1 6 0.2,respectively. In addition, a new group, pKa 9.3 6 0.2, appeared,deprotonation of which increased kcat/Km

NADPH nearly 2-fold(Fig. 2F).

The Cys630 substitution did not eliminate the acidic pKa butshifted it to 7.8 6 0.1 for both the kcat and kcat/Km

NADPH profiles(Fig. 2, G and H), suggesting that, although Cys630 is not theionizing residue, it does influence the pKa of this group. TheC630A mutant exhibited the same behavior as the Asp675 andSer457 mutants in the basic limb of the pH profile, displaying ashift to a higher pKa. In general, the increase in the basic pKa

was observed in all mutants with decreased catalytic activity(data for other mutants not shown), suggesting that this shiftmay arise from a change in the rate-limiting step for cyto-chrome c reduction.

Flavin Reduction—The kinetics of reduction of the mutantP450R proteins by NADPH were examined by monitoring ab-sorbance changes at 452 nm, associated with flavin reduction,and 585 nm, associated with semiquinone formation (34–38).Fig. 3 shows the absorbance changes associated with NADPHreduction at low ionic strength (50 mM Tris, pH 7.7, I1/2 5 35mM) for the wild-type enzyme as well as the C630A, S457A, andD675N mutants, with calculated rate constants given in TableIII. At low ionic strength, aerobic NADPH reduction of thewild-type enzyme was biphasic, with an initial fast phase, k1 555 s–1, accounting for about 80% of the absorbance change,followed by a slower phase, k2 5 4.0 s21, accounting for about20% of the absorbance change (18). These values are compara-ble with those obtained with pig and rabbit reductases underanaerobic conditions (35, 36). An isotope effect of 3.4 was ob-served for the first phase of wild-type NADPH reduction, whileno isotope effect was observed for the second phase (18). Mu-tation of residue Ser457, Asp675, or Cys630 produced large de-creases in overall reduction rates, with varying effects on therates and amplitudes of the fast and slow phases (Fig. 3, A andC, and Table III). A large deuterium isotope effect has previ-ously been observed for NADPH reduction of the S457A mutant(18). Similarly, large deuterium isotope effects were also ob-served for the Asp675 and Cys630 mutants; these were associ-ated with the phase having the larger A452 change, suggestingthat hydride transfer occurred in this phase.

Reduction of the D675N mutant by NADPH under anaerobicconditions resembled that of wild-type enzyme, with 84% of thetotal absorbance change and the isotope effect occurring in thefirst phase. Rate constants were decreased 50- and more than500-fold, respectively, for the first and second phases of reduc-tion (Fig. 3A and Table III). In contrast, NADPH reduction offour other mutants (D675V, S457A/D675N, C630A, and S457A/

TABLE IIKinetic properties of active-site mutants

Reactions were carried out in 0.27 M potassium phosphate and contained 65 mM cytochrome c (for KmNADPH) and 50 mM NADPH (for Km

cyt c) andvarying amounts of NADPH or cytochrome c. Reactions were initiated by the addition of protein after a 2-min preincubation at 28 °C. Values areexpressed as mean 6 S.D. (n).

Protein kcat KmNADPH Km

cyt c KiNADP1 DVa D(V/K)b

min21 mM mM mM

Wild type 5421 6 746 (4) 6.2 6 0.7 (4) 16.3 6 1.7 (6) 18.8 6 1.9 (3) 2.7 (2) 2.1 (2)S457Ac 60 6 6 (5) 6.3 6 0.5 (5) ,1 (3) 18.2 6 2.1 (4) 9.0 6 0.4 (3) 9.0 6 0.4 (3)D675N 310 6 25 (3) 5.8 6 0.3 (3) 2.7 6 0.1 (3) 7.6 6 1.7 (3) 6.9 6 0.6 (3) 6.0 6 2.3 (3)D675V 8 6 1 (3) 5.4 6 0.2 (3) ,1 (3) 27.5 6 3.8 (3) 7.7 6 0.5 (3) 4.2 6 0.8 (3)D675N/S457A 7 6 1 (3) 6.0 6 1.5 (3) ,1 (3) 14.0 6 2.4 (3) 7.8 (2) 9.3 (2)C630A 110 6 26 (3) 5.0 6 1.0 (3) ,1 (3) 7.3 (1) 9.8 6 1.0 (3) 10.4 6 2.1 (3)S457A/D675N/C630A ;0.24 (3) NDd ND ND ND ND

a Primary deuterium isotope effect on kcat. Reactions were carried out as described under “Materials and Methods.”b Primary deuterium isotope effect on kcat/Km

NADPH. Reactions were carried out as described under “Materials and Methods.”c Taken from Ref. 18.d ND, not determined.

FIG. 2. pH dependence of specific activity, kcat, and kcat/Km

NADPH for wild-type and mutant P450R proteins. A, wild-typekcat; B, S457A specific activity; C, D675N kcat; D, D675N kcat/Km

NADPH;E, D675V kcat; F, D675V, kcat/Km

NADPH; G, C630A kcat; H, C630Akcat/Km

NADPH.

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D675N/C630A) differed in that most of the A452 change and theisotope effect occurred in the second phase of reduction (k2)rather than the first. In the case of the D675V and S457A/D675N mutants, .80% of the total A452 changes were associ-ated with the second phase of reduction (Fig. 3C and Table III),as was the isotope effect. Replacement of Cys630 with alanineproduced approximately 20-fold decreases in both k1 and k2,with 80% of total absorbance change associated with k2 (Fig. 3Aand Table III). The isotope effect was also associated primarilywith k2. Substitution of all three residues resulted in an ex-tremely slow rate of reduction by NADPH, characterized by atransient initial phase, followed by a second phase which ex-hibited the isotope effect and 93% of the total A452 change (Fig.3E and Table III). k2 for this second phase was 104-fold lowerthan wild-type k2 and 5 orders of magnitude lower thanwild-type k1.

Formation of wild-type flavin semiquinone as monitored byabsorbance changes at 585 nm was concomitant with flavinreduction, suggesting rapid interflavin electron transfer (34–38). S457A semiquinone formation was biphasic, with an initialrate comparable with that of flavin reduction (Fig. 3D andTable III; Ref. 18). A585 changes for the D675N and S457/D675N mutants, however, were small and slower than thecorresponding rates of flavin reduction (Fig. 3, B and D, TableIII). The A585 changes observed for the D675V, C630A, andS457A/D675N/C630A mutants were biphasic, with a transientfirst phase followed by a second, slower phase with rate con-stants comparable with the second, slow phase of flavin reduc-tion (Fig. 3, B and F; Table III).

Anaerobic NADPH Titration of P450R—Anaerobic NADPHtitration was used to investigate the effects of the Asp675 andCys630 substitutions on flavin redox potential. Titration of ratliver P450R with NADPH under anaerobic conditions has been

shown to produce visible absorption spectra that are charac-teristic for each redox state of the enzyme (31, 39). The additionof 0.5 mol of NADPH/mol of enzyme to the fully oxidized pro-tein produces the spectrum of the air-stable semiquinone (FAD/FMNHz), characterized by an absorbance decrease at 452 nm,isosbestic points at 363 and 502 nm, and the appearance of along wavelength absorbance with a maximum at 585 nm and ashoulder at 630 nm. The further addition of NADPH producesthe 3e2-reduced form (FMNH2/FADHz), with a further absorb-ance decrease at 452 nm, minimal absorbance changes at 585nm, and a slightly altered long wavelength absorbance (lmax at593 nm and shoulder at 630 nm) characteristic of the FADsemiquinone (FADHz). Since the midpoint potential of theFADHz/FADH2 couple is below that of NADPH, NADPH isunable to reduce P450R to the 4e2-reduced stage (31, 39) andthe further addition of NADPH to the 3e2-reduced enzymeproduces minimal absorbance decreases at 452 nm. Identicalabsorbance changes are seen upon NADPH titration of therecombinant enzyme, while the S457A titration produces spec-tra consistent with a decrease in the FAD/FADHz redoxpotential (18).

Fig. 4 presents the absorbance changes at 452 and 585 nmobserved upon the addition of NADPH under anaerobic condi-tions to the wild-type and mutant proteins. Reduction of theD675N protein to the 1e2-reduced (FAD/FMNHz) stage paral-leled that seen for the wild-type enzyme (Fig. 4, A and C), withproduction of the air-stable semiquinone upon the addition ofapproximately 0.5 mol of NADPH/mol of protein. However, thefurther addition of NADPH to the D675N protein producedminimal changes in A452 but a decrease in A585, consistent withreduction of FMNHz to FMNH2 and no formation of FADHz.This behavior is similar to that reported previously for theS457A mutant and suggests that the D675N mutation, like theS457A mutation, decreases the FAD/FADHz redox potential. Incontrast, the S457A/D675N (Fig. 4D) double mutant requirednearly 2 mol of NADPH/mol of protein for full production of the1e2-reduced protein, with no further absorbance changes ateither wavelength observed upon further addition of NADPH,consistent with a decreased FAD/FADH2 redox potential.

Reduction of the D675V mutant to the 1e2-reduced stagerequired approximately twice as much NADPH as the wild-type protein (Fig. 4B) and the further addition of NADPHproduced no further decreases in A452 and a slight decrease inA585, consistent with a decreased FADHz/FADH2 redox poten-tial. NADPH reduction of the C630A mutant proceeded in amanner similar to wild type (Fig. 4E), although the magnitudeof the decrease in A452 was slightly smaller than that of wildtype. Finally, formation of the 1e2-reduced form of the S457A/D675N/C630A protein required more than 4 mol of NADPH/mol of protein, and A585 but not A452 decreased upon furtheraddition of NADPH, suggesting that both the FAD/FADHz andFAD/FADH2 redox potentials were decreased (Fig. 4F).

DISCUSSION

We have previously shown that Ser457 is a residue crucial forhydride transfer and control of FAD redox potential (18). Thepresent studies demonstrate that Cys630 and Asp675 of P450Rare also located in the active site for FAD reduction and have akey role in efficient hydride transfer from NADPH to FAD;removal of all three residues abolishes catalytic activity. Cys630

and Asp675, like Ser457, function primarily in catalysis andhave minimal effects on NADPH and NADP1 binding andflavin content. The decrease in Km

cyt c concomitant with thedecrease in kcat is consistent with the nonclassical two-siteping-pong mechanism proposed for P450R (32). For each of theactive site mutants examined, the decreases in kcat parallelchanges in the rate of flavin reduction and are associated with

FIG. 3. Kinetics of anaerobic reduction of mutant P450R pro-teins by NADPH. Protein solutions were made anaerobic as describedunder “Materials and Methods,” and NADPH was added to give a 5:1ratio of NADPH to protein. A452 values are normalized to 1 for compar-ison purposes. Lines are fits of data points to Equation 6 or 7. A and Bshow A452 and A585 changes for wild-type (●), D675N (E), and C630A(Œ) proteins. C and D show A452 and A585 changes for D675V (●),S457A/D675N (E), and S457A (Œ). E and F show A452 and A585 changesfor S457A/D675N/C630A.

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large NADPD isotope effects, indicating that the effects of thesesubstitutions on catalysis are primarily a result of impairedhydride transfer. Activities of multiply-substituted reductasemutants were roughly multiplicative, strongly suggesting thatthese residues interact to facilitate hydride transfer.

Comparison of the effects of the D675V and D675N substi-tutions suggests that hydrogen bond formation between Asp675

and some other group, probably Ser457, is necessary for hydridetransfer. kcat for the D675N mutation, which retains hydrogenbonding capacity, is decreased only 14-fold, while eliminationof hydrogen bonding interactions with the D675V and S457A/D675N mutants decreases kcat 700-fold. The ionic strengthdependence of cytochrome c reduction is also shifted to higherionic strengths by the D675N mutation (data not shown), con-sistent with the presence of a stronger hydrogen bond with thismutant. Although Ser457 and Asp675 do interact to influencehydride transfer, the effect of removing the Ser457 hydroxylgroup (S457A) is 10-fold less than that of removal of the Asp675

carboxyl group (D675V), perhaps because the S457A mutation

allows positioning of a water molecule in the active site, whichcould partially restore activity. Alternatively, the D675V sub-stitution could interfere with some other step in addition tohydride transfer, such as formation of the enzyme-NADPHcomplex or NADP1 release. X-ray crystallography of FNR in-dicates that residues Glu312 and Ser96, homologous to Asp675

and Ser457 of P450R, are within hydrogen bonding distance(2.7 Å) of each other, with Ser96 possibly interacting with theN-5 of FAD (20). The P450R crystal structure indicates thatSer457 is within hydrogen bonding distance of the flavin N-5,but hydrogen bonding between Ser457 and Asp675 has not beenestablished (15).

Previous results have demonstrated dependence of wild-typekcat on a macroscopic pKa of approximately 6.9 (21). This mac-roscopic pKa could reflect deprotonation of a single functionalgroup, which in turn could be influenced by other ionizablegroups in the microenvironment of the protein. Elimination orperturbation of this pKa by mutagenesis provides a means ofinvestigating the group(s) involved. Results from this studyshow that, although Asp675 is necessary for catalysis, its pKa

does not fall between 5.5 and 10, and some other group isresponsible for the 6.9 pKa. This unknown acidic group isinfluenced by Cys630; removal of the sulfhydryl increases thepKa by nearly 1 pH unit. Since catalytic activity is virtuallyabolished by the S457A/D675N/C630A triple mutation, it isunlikely that there is another, unidentified catalytic aminoacid. A candidate for this acidic group, then, is the N-1 of FAD,whose pKa in the unbound state is 6.3 (40). Donation of a protonfrom Cys630 could stabilize the reduced FADH2, thereby in-creasing the redox potential and facilitating hydride transfer;this is consistent with the decreases in FAD redox potentialand rate of hydride transfer observed with the C630A mutant.

The Asp675 mutations as well as the C630A, S457A, andother carboxyl-terminal mutations2 all shift the pKa of thebasic group from 8.8 to .10. This may be due to a shift in therate-limiting step from this pH-dependent step to the hydridetransfer step.

Reduction of P450R by NADPH proceeds through rapid for-mation of one or more charge-transfer complexes (35, 41), fol-lowed by slower hydride transfer to FAD; hydride transfer islargely rate-limiting for cytochrome c reduction (32). Biphasicabsorbance changes at 452 and 585 nm are observed uponNADPH reduction of wild-type reductase. The first phase oc-curs at a rate consistent with enzyme turnover and correspondsto FAD reduction and rapid interflavin electron transfer, whilethe second, slow phase has been proposed to correspond to

2 A. L. Shen and C. B. Kasper, unpublished observations.

TABLE IIIReduction of active-site mutants by NADPH

Reactions were carried out in 50 mM Tris, pH 7.7, at 20 °C, and data were fitted to Equation 6 (monophasic) or Equation 7 (biphasic) to yield rateconstants k1 and k2 for reduction. Data were obtained from at least two separate protein preparations and are expressed as mean 6 S.D. (n). Thewild-type protein was reduced under aerobic conditions. The mutant proteins were reduced under anaerobic conditions.

ProteinA452 A585

k1 k2Fast phasereductiona Db k1 k2

s21 s21 % s21 s21

Wild typec 55 6 3.3 (9) 4.05 6 0.48 (9) 80 3.4 (k1) 51.4 3.4S457A 0.31 6 0.03 (4) 100 .20 (k1) ,1.3 0.03D675N 1.4 6 0.2 (5) ,0.007 (5) 84 .20 (k1) 0.02 6 0.05 (5)D675V 1.4 6 0.3 (4) 0.05 6 0.001 (4) 15 .20 (k2) 0.1 6 0.2 (4) 0.03 6 0.01 (4)C630A 2.6 6 0.8 (7) 0.2 6 0.02 (7) 20 .20 (k2) 0.7 6 0.04 (4) 0.1 6 0.04 (4)S457A/D675N 0.76 6 0.24 (3) 0.05 6 0.003 (3) 10 .20 (k2) 0.002 6 0.02 (3)S457A/D675N/C630A 0.11 6 0.06 (5) 0.0005 6 0.00005 (5) 7 .20 (k2) 0.01 6 0.004 (5) 0.0005 6 0.0001 (5)

a Percentage of reduction occurring in fast phase (rate constant k1).b Deuterium isotope effect on rate of flavin reduction. The rate constant with which the isotope effect is associated is shown in parentheses.c Taken from Ref. 18.

FIG. 4. NADPH titration of wild-type and mutant P450R pro-teins under anaerobic conditions. Absorbance changes at 452 (●)and 585 (E) nm are plotted versus mol of NADPH added/mol of protein.A, wild type, 27 mM; B, D675V, 12 mM; C, D675N, 7 mM; D, S457A/D675N, 12 mM; E, C630A, 18 mM; F, S457A/D675N/C630A, 18 mM.

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reduction of the 2-electron-reduced reductase by a second mol-ecule of NADPH and is limited by the rate of NADP1 releaseand/or some conformational change (35). Consistent with thishypothesis, a deuterium isotope effect for reduction of the wild-type enzyme by NADPD is observed only for the first phase butnot the second (18).

Like the wild-type enzyme, reduction of the D675N proteinby NADPH produces biphasic kinetics. The rate of the firstphase, k1, is consistent with kcat (Table III), while the rate forthe second phase is slower than that of overall turnover andshows no deuterium isotope effect. In contrast, althoughNADPH reduction of the D675V, S457A/D675N, and S457A/D675N/C630A mutants is also biphasic, the rate of the second,rather than the first, phase exhibits the isotope effect. Thissuggests that the second phase, with rate constant k2, repre-sents hydride transfer, and k1 may represent some prior step,perhaps the formation of a P450R-NADPH pre-hydride trans-fer complex. Pre- and post-hydride transfer complexes havebeen observed spectroscopically for other members of this fam-ily, such as FNR (42), phthalate dioxygenase reductase (43),and P450-BM3 (44), and postulated, but never observed, in thecase of P450R (35, 41). The greatly decreased rate of hydridetransfer and, perhaps, alterations in the conformation of theNADPH-FAD complex allow observation of the pre-hydridetransfer complex in these proteins.

Results of the anaerobic NADPH titrations indicate thatthese substitutions modulate FAD redox potential (Fig. 4; Ref.18). The D675N and S457A/D675N/C630A mutants, like theS457A mutant, destabilize the FADHz semiquinone, while theD675V and S457A/D675N (and possibly the Cys630) mutationsdecrease the FADHz/FADH2 redox potential.

While rates of semiquinone formation are roughly compati-ble with kcat for cytochrome c reduction for the wild-type,S457A, C630A, D675V, and S457A/C630A/D675N proteins(when temperature and ionic strength differences are ac-counted for), semiquinone formation for the D675N and S457A/D675N mutants is slower than kcat. A similar phenomenonoccurs with FNR; recently, Medina et al. (45) have demon-strated destabilization of the FADH semiquinone and inhibi-tion of activities requiring 1e2 transfer in the Glu301 mutant ofFNR, which is homologous to Asp675.

A model for hydride transfer consistent with the kinetic datais presented in Fig. 5. Ser457 stabilizes the reduced flavin byaccepting a hydrogen bond from the reduced N-5. Asp675 hy-drogen bonds to Ser457, orienting Ser457 and/or making it astronger hydrogen bond acceptor. These residues may also actto position the flavin and nicotinamide in an orientation suit-able for hydride transfer. Cys272 of FNR, which is equivalent toCys630 of P450R, is located about 6 Å from the flavin N-5, andmodeling studies propose that it may contact the nicotinamidering (12). We propose that Cys630 interacts with both the FADand the nicotinamide and may catalyze hydride transfer bydestabilizing the nicotinamide C-4–H bond and/or donating aproton to the reduced flavin. In this position, it may also be ableto accept a proton from the anionic FAD semiquinone (FADH–)after electron transfer to FMN to yield the observed blue neu-tral semiquinone.

Although the residues corresponding to Ser457, Cys630, andAsp675 are conserved in all members of the FNR family andmutagenesis of corresponding residues of FNR, Ser96 andCys272, decreases FNR catalytic activity and FAD redox poten-tial (46, 47), the active sites of these proteins are designed forthe different functions carried out by these enzymes: NADPHoxidation coupled to substrate reduction in P450R, nitric-oxidesynthase, and P450BM3 versus NADPH production in FNR.The rate and direction of hydride transfer between NADPH

and the N-5 of the flavin isoalloxazine depend both on therelative redox potentials and the orientation of the flavin andpyridine rings in some optimum geometry. The FAD redoxpotential is dependent upon the stabilization of the anionicreduced flavin, which is influenced by hydrogen bonding inter-actions between the protein and N-1, O-2, O-4, and N-5 of theflavin. The large isotope effects obtained with these mutantsare suggestive of the presence of tunneling in the hydridetransfer, which requires a precise geometry as well as a shortdistance between the flavin N-5 and nicotinamide C-4. It ap-pears that the relative positions of these residues are critical indetermining both the rate and equilibrium of the hydridetransfer reaction; crystallographic analysis of these mutantsmay identify the hydrogen bonding patterns important for hy-dride transfer and control of flavin redox potential.

Acknowledgments—We are grateful to Timothy Culligan and JohnSheehan for technical assistance with protein purification, to Dr. BrianFox and Dr. Dexter Northrop for use of the stopped-flow spectropho-tometers and for helpful discussions, and to Mary Jo Markham andKristen Adler for preparation of this manuscript.

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