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

Anna L. Shen, Daniel S. Sem, and Charles B. KasperDagger

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

    ABSTRACT
Top
Abstract
Introduction
References

Site-directed mutagenesis has been employed to study the mechanism of hydride transfer from NADPH to NADPH-cytochrome P450 oxidoreductase. Specifically, Ser457, Asp675, and Cys630 have been selected because of their proximity to the isoalloxazine ring of FAD. Substitution of Asp675 with asparagine or valine decreased cytochrome c reductase activities 17- and 677-fold, respectively, while the C630A substitution decreased enzymatic activity 49-fold. Earlier studies had shown that the S457A mutation decreased cytochrome c reductase activity 90-fold and also lowered the redox potential of the FAD semiquinone (Shen, A., and Kasper, C. B. (1996) Biochemistry 35, 9451-9459). The S457A/D675N and S457A/D675N/C630A mutants produced roughly multiplicative decreases in cytochrome c reductase activity (774- and 22000-fold, respectively) with corresponding decreases in the rates of flavin reduction. For each mutation, increases were observed in the magnitudes of the primary deuterium isotope effects with NADPD, consistent with decreased rates of hydride transfer from NADPH to FAD and an increase in the relative rate limitation of hydride transfer. Asp675 substitutions lowered the redox potential of the FAD semiquinone. In addition, the C630A substitution shifted the pKa of an ionizable group previously identified as necessary for catalysis (Sem, D. S., and Kasper, C. B. (1993) Biochemistry 32, 11539-11547) from 6.9 to 7.8. These results are consistent with a model in which Ser457, Asp675, and Cys630 stabilize the transition state for hydride transfer. Ser457 and Asp675 interact to stabilize both the transition state and the FAD semiquinone, while Cys630 interacts with the nicotinamide ring and the fully reduced FAD, functioning as a proton donor/acceptor to FAD.

    INTRODUCTION
Top
Abstract
Introduction
References

The microsomal and nuclear envelope flavoprotein NADPH-cytochrome P450 oxidoreductase (P450R1; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4; hereafter referred to as reductase), one of a family of FMN- and FAD-containing enzymes that includes nitric-oxide synthase, the sulfite reductase alpha -subunit, and P450BM-3 (for recent reviews, see Refs. 1 and 2), mediates the transfer of electrons from NADPH to cytochrome P450 and other microsomal proteins as well as to nonphysiological electron acceptors such as cytochrome c and ferricyanide (3). Electron transfer to electron acceptors such as cytochromes c or P450 proceeds from NADPH to FAD to FMN, while ferricyanide and 3-acetylpyridine adenine dinucleotide phosphate (AcPyrADP) accept electrons directly from FAD (4-6).

Sequence comparisons as well as a variety of biochemical studies (6-11) have suggested the presence of independent FMN- 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 to any known protein from the FAD/NADPH binding domain, which is related to another class of flavoproteins, the transhydrogenases (1, 2, 12), and includes ferredoxin-NADP+ reductase (FNR), NADH-nitrate reductase, and NADH-cytochrome b5 reductase as well as phthalate dioxygenase reductase (13). Both FNR and P450R abstract the pro-R (A-side) hydrogen of NADPH (12, 14), with the conformation of the bound nicotinamide anti in P450R (14) but unknown for other members of this family. These sequence similarities have been confirmed by the recently described x-ray crystal structure of P450R, which also highlights the role of the insertion sequence in aligning the flavin isoalloxazine rings in a position for direct electron transfer (15). Finally, potential flavin- and NADPH-binding, as well as catalytic, residues identified by sequence comparisons of P450R with flavodoxin and FNR (2, 7) have been confirmed by biochemical studies, including site-directed mutagenesis and kinetic measurements (16-18), as well as crystallographic studies (15).

The crystal structures of FNR and P450R place three conserved residues, Ser457, Cys630, and Asp675, in close proximity to the isoalloxazine ring of FAD. Ser457 is in a position to form a hydrogen bond with the oxidized or reduced flavin N-5 of P450R, while the homologous Ser96 of FNR interacts with the reduced, but not the oxidized, flavin N-5, suggesting a role for these residues in hydride transfer and/or stabilization of the reduced flavin (12, 15, 19, 20). We have previously shown that substitution of Ser457 produces large decreases in the rate of hydride transfer from NADPH to FAD and lowers the FAD/FADH ·redox potential (18). pH studies have indicated that catalysis is dependent upon deprotonation of an acidic group having a pKa between 6.2 and 7.3 (21); comparison with FNR suggests that Asp675 could fill this role (2, 12). The current study explores the roles of Cys630 and Asp675 of NADPH-cytochrome P450 oxidoreductase in catalysis and FAD reduction and shows that the three residues Ser457, Asp675, and Cys630 interact to form the catalytic site for hydride transfer from NADPH to FAD.

    MATERIALS AND METHODS

Expression and purification of recombinant NADPH-cytochrome P450 oxidoreductase was carried out as described previously (16), except that cultures were grown and induced at 28 instead of 37 °C. Protein was assayed by the BCA method (22), and FMN and FAD were determined by the method of Faeder and Siegel (23). Cytochrome c, ferricyanide, and AcPyrADP activities were assayed as described previously (24), except that reactions were initiated by the addition of protein. Steady-state kinetic parameters were determined in the presence of 0.27 M potassium phosphate, pH 7.7.

The D675N and C630A mutants were constructed by the method of Kunkel (25), using the following 5'-oligonucleotides: D675N, ACTCACTAAACGTGTGGAGC; C630A, TCTATGTGGCCGGGGATGC. The D675V mutant was prepared by PCR (26), using the following oligonucleotides: D675V, GGGTCCTAGGTCCTAGCTCCACACAACTAGTGAGTA; 1858, GTGTGAGCTGCTGCCACGCC. The D675V oligonucleotide contains the indicated mutation and an AvrII site for cloning of the mutant fragment. PCR reactions contained 10 mM Tris, pH 8.3, 50 mM KCl, 6.5 mM MgCl2, 0.001% gelatin, 200 µM of each dNTP, 10 µg/ml template DNA, 1 µM D675V mutagenic oligonucleotide, 1 µM 1858 oligonucleotide (hybridizing to bases 5' of the mutation site), and 0.05 units AmpliTaq polymerase (Perkin-Elmer, Foster City, CA). Reaction conditions were as follows: 94 °C, 1 min; 47 °C, 1 min; 72 °C, 2 min; 25 cycles, followed by extension for 5 min at 72 °C. Ampliwax (Perkin-Elmer) was used according to the manufacturer's instructions. After removal of free oligonucleotides by centrifugation through an Mr 100,000 cut-off filter (Millipore Corp., Bedford, MA), PCR-amplified DNA fragments were digested with AvrII and NheI, purified by agarose gel electrophoresis, and cloned into pOR263. All mutant plasmids were characterized by restriction mapping and sequencing of the PCR-amplified regions and cloning sites. Sequencing was performed with the AmpliTaq cycle sequencing kit (Perkin-Elmer). Multiply substituted mutants were constructed by restriction enzyme digestion and cloning of the mutant fragments into pOR263 and confirmed by sequencing.

For studies of the primary deuterium isotope effect, A-side NADPD was synthesized and purified by high performance liquid chromatography as described previously (18). The fraction of deuteration was determined as described previously (27) and ranged from 0.8 to 0.9. Cytochrome c assays for isotope effect studies were carried out in 10 mM potassium phosphate, 0.45 M KCl, pH 7.7 (I1/2 = 478 mM). Primary deuterium isotope effects were determined by the method of direct comparison (28) and fitted to the following equation, which assumes different isotope effects on Vmax and Vmax/KmNADPH (27, 28),
v=<FR><NU>V*A</NU><DE>K<SUB>m</SUB>*(1+F<SUB>i</SUB>*E<SUB>V/K</SUB>)+A*(1+F<SUB>i</SUB>*E<SUB>v</SUB>)</DE></FR> (Eq. 1)
where v is the initial velocity, A is the concentration of NADPH, Fi is the fraction of deuterium label, EV/K is the isotope effect minus 1 on Vmax/KmNADPH, 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, and 11, were carried out in CAPS buffer. Data were fitted to Equations 2-5 (30) as follows,
Y=<FR><NU>Y<SUB>0</SUB></NU><DE>1+(H/K<SUB>1</SUB>)+(K<SUB>2</SUB>/H)</DE></FR> (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 their preferred ionization state,
Y=<FR><NU>Y<SUB>H</SUB></NU><DE>1+(H/K<SUB>1</SUB>)+(K<SUB>2</SUB>/H)</DE></FR>+<FR><NU>Y<SUB>L</SUB></NU><DE>(K<SUB>1</SUB>/H)+1</DE></FR> (Eq. 3)
Y=<FR><NU>(Y<SUB>L</SUB>*(H/K<SUB>1</SUB>))+Y<SUB>H</SUB></NU><DE>(H/K<SUB>1</SUB>)+1</DE></FR> (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=<FR><NU>Y<SUB>M</SUB></NU><DE>1+(H/K<SUB>1</SUB>)+(K<SUB>2</SUB>/H)</DE></FR>+<FR><NU>Y<SUB>H</SUB></NU><DE>1+(H/K<SUB>3</SUB>)+(K<SUB>2</SUB>/H)</DE></FR> (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 acidic groups, and K2 is the acid dissociation constant for the basic group.

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

Measurements of visible absorption spectra and anaerobic NADPH reduction rates and anaerobic NADPH titrations were carried out at 28 °C on a Beckman 7500 diode array spectrophotometer. Anaerobic reactions contained 50 mM Tris, pH 7.7, 10 mM glucose, 5u/ml glucose oxidase, 1 µM methyl viologen, and P450R at the concentrations indicated (31). Cuvettes were sealed, and oxygen was removed by subjecting the samples to several cycles of evacuation followed by flushing with argon purified by passage through a scrubbing tower containing 0.5% dithionite, 0.1% 2-anthroquinone sulfonate, and 0.4% NaOH. Aerobic stopped-flow studies were carried out on an Olis RSM 100 rapid scanning spectrophotometer. Reactions were carried out in 50 mM Tris, pH 7.7, 28 °C, and data were fitted to the following equations,
A<SUB>t</SUB>=A<SUB>0</SUB>*e<SUP>(<UP>−</UP>k<SUB>1</SUB>*t)</SUP> (Eq. 6)
A<SUB>t</SUB>=(A<SUB>0</SUB>*e<SUP>(<UP>−</UP>k<SUB>1</SUB>*t)</SUP>)+(A<SUB>1</SUB>*e<SUP>(<UP>−</UP>k<SUB>2</SUB>*t)</SUP>) (Eq. 7)
where At is the absorbance at time t, and A0 and A1 are the initial absorbance values for the first and second phases of reduction, respectively.

    RESULTS

Spectral Properties-- Fig. 1 shows the visible absorption spectra of the oxidized wild-type and mutant reductases, with broad peaks at approximately 380 and 452 nm similar to that described for the rat liver enzyme (4). The addition of NADPH under aerobic conditions to each of the proteins produced spectra characteristic of the air-stable semiquinone (FAD/FMNH·), with a decreased absorbance at 452 nm and a long wavelength absorbance band having a maximum at approximately 585 nm and a shoulder at 630 nm. None of the substitutions produced significant changes in the visible absorption spectra. This is in agreement with results of flavin analyses, which indicated no effect of any of the substitutions on FMN or FAD content.


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Fig. 1.   Visible absorption spectra of wild-type and mutant P450R proteins. Semiquinone spectra were obtained by adding NADPH under aerobic conditions and recording after equilibrium had been reached (>15 min). A, wild type, 32 µM; B, D675V, 18 µM; C, D675N, 13 µM; D, C630A, 20 µM; E, S457A/D675N, 11 µM; F, S457A/D675N/C630A, 22 µM. Solid lines, oxidized; broken lines, semiquinone.

Enzymatic Activity-- Substitution of any one of the three residues Ser457, Asp675, or Cys630 produced large decreases in rates of reduction of cytochrome c, which accepts electrons from FMN, and ferricyanide and AcPyrADP, each of which accepts electrons from FAD, suggesting that these mutations interrupt electron transfer from NADPH to FAD. Specific activities in the presence of 0.27 M potassium phosphate, pH 7.7, and saturating substrate concentrations are shown in Table I. The largest decrease in cytochrome c reductase activity was produced by the D675V substitution, which had only 0.1% of wild-type activity. Activities with the substrates ferricyanide and AcPyrADP were decreased 85- and 39-fold, respectively. The D675N substitution, which retains hydrogen-bonding capacity, produced smaller effects, with only a 14-fold decrease in cytochrome c reductase activity and 15- and 5-fold decreases, respectively, in activities with the substrates ferricyanide and AcPyrADP. Substitution of Cys630 with alanine produced a 37-fold decrease in cytochrome c reductase activity and 30- and 13-fold decreases, respectively, in electron transfer to ferricyanide and AcPyrADP.

                              
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Table I
Specific activities of active-site mutants

We have previously reported a 90-fold decrease in cytochrome c reductase activity as a result of the S457A substitution (18). Combination of the S457A mutation with substitutions at Asp675 and Cys630 produced multiplicative effects on cytochrome c reductase activities. Cytochrome c reductase activity of the S457A/D675N double mutant was decreased ~600-fold, while cytochrome c reductase activity of the S457A/D675N/C630A triple mutant was very close to background levels, with a calculated specific activity more than 4 orders of magnitude less than wild type. Ferricyanide reductase activity of the S457A/D675N mutant was decreased to 6% of wild type and AcPyrADP activity to 1% of wild type.

Steady-state Kinetics-- Table II shows steady-state kinetic parameters for the active-site mutants, with cytochrome c as electron acceptor. As was demonstrated previously for the Ser457 mutations (18), substitutions of Asp675 and Cys630 produced large decreases in kcat, with no significant changes in KmNADPH, indicating that these mutations affect catalysis only and not cofactor binding. As with the wild-type and S457A enzymes, NADP+ was a competitive inhibitor versus NADPH; KiNADP+ was only minimally affected by substitutions of Asp675 and Cys630. Kmcyt c values decreased in parallel with the decreases in kcat, consistent with the nonclassical two-site ping-pong mechanism of P450R (32) and a decrease in the rate of the reductive half-reaction (33).

                              
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Table II
Kinetic properties of active-site mutants
Reactions were carried out in 0.27 M potassium phosphate and contained 65 µM cytochrome c (for KmNADPH) and 50 µM NADPH (for Kmcyt c) and varying amounts of NADPH or cytochrome c. Reactions were initiated by the addition of protein after a 2-min preincubation at 28 °C. Values are expressed as mean ± S.D. (n).

To assess the relative rate limitation of hydride transfer for the active-site mutants, the deuterium isotope effect on cytochrome c reduction with [(4S-H),(4R-D)]NADPD was determined. 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 observed on Vmax/KmNADPH, consistent with hydride transfer being partially rate-limiting. Substitution of Asp675 increased the isotope effects on Vmax and Vmax/KmNADPH to values ranging from 4 to 7 (Table II). Although the activity of the C630A mutant was about 10% of wild type, the deuterium isotope effects on both Vmax and Vmax/KmNADPH were increased markedly, to approximately 10. The double substitution, D675N/S457A, produced isotope effects similar to those of the S457A single mutant.

pH Dependence of Cytochrome c Reductase Activity-- The pH dependence of wild-type kcat and kcat/KmNADPH has been previously shown to be dependent upon two ionizable groups. The first has a pKa of 6.2 at 850 mM ionic strength and 7.1 at 350 mM ionic strength, which must be unprotonated for maximum activity, while the other has a pKa of approximately 8.8, which must be protonated for maximum activity (21). To determine if these pKa values could be attributed to one or more of these active-site residues, the pH dependence of kcat and kcat/KmNADPH with cytochrome c as the substrate was determined.

Fig. 2 shows the pH dependence of kcat and kcat/KmNADPH for the D675N, D675V, S457A/D675N, and C630A mutants. The pH dependences of wild-type kcat and S457A cytochrome c reductase activity are also presented. At 475 mM ionic strength, wild-type kcat displayed pKa values of 6.9 ± 0.1 and 9.6 ± 0.1 (Fig. 2A), consistent with previous results (29). The S457A mutant (Fig. 2B) displayed the same acidic pKa (6.8), but the pKa 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 is responsible for this pKa. The pH dependence of kcat and kcat/KmNADPH for the D675N mutant (Fig. 2, C and D) was similar to that of wild type in the acidic limb, with acidic pKa values of 7.1 ± 0.2 and 6.8 ± 0.3, respectively, but the basic pKa values were again shifted to >10. D675V kcat showed only a 2-fold change over the pH range assayed; however, curve fitting indicated the presence of an acidic pKa of 6.6 ± 0.2 and an increase 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 ± 0.2 and 10.1 ± 0.2, respectively. In addition, a new group, pKa 9.3 ± 0.2, appeared, deprotonation of which increased kcat/KmNADPH nearly 2-fold (Fig. 2F).


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Fig. 2.   pH dependence of specific activity, kcat, and kcat/KmNADPH for wild-type and mutant P450R proteins. A, wild-type kcat; B, S457A specific activity; C, D675N kcat; D, D675N kcat/KmNADPH; E, D675V kcat; F, D675V, kcat/KmNADPH; G, C630A kcat; H, C630A kcat/KmNADPH.

The Cys630 substitution did not eliminate the acidic pKa but shifted it to 7.8 ± 0.1 for both the kcat and kcat/KmNADPH profiles (Fig. 2, G and H), suggesting that, although Cys630 is not the ionizing residue, it does influence the pKa of this group. The C630A mutant exhibited the same behavior as the Asp675 and Ser457 mutants in the basic limb of the pH profile, displaying a shift 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 shift may arise from a change in the rate-limiting step for cytochrome c reduction.

Flavin Reduction-- The kinetics of reduction of the mutant P450R proteins by NADPH were examined by monitoring absorbance 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 NADPH reduction at low ionic strength (50 mM Tris, pH 7.7, I1/2 = 35 mM) for the wild-type enzyme as well as the C630A, S457A, and D675N mutants, with calculated rate constants given in Table III. At low ionic strength, aerobic NADPH reduction of the wild-type enzyme was biphasic, with an initial fast phase, k1 = 55 s-1, accounting for about 80% of the absorbance change, followed by a slower phase, k2 = 4.0 s-1, accounting for about 20% of the absorbance change (18). These values are comparable with those obtained with pig and rabbit reductases under anaerobic conditions (35, 36). An isotope effect of 3.4 was observed for the first phase of wild-type NADPH reduction, while no isotope effect was observed for the second phase (18). Mutation of residue Ser457, Asp675, or Cys630 produced large decreases in overall reduction rates, with varying effects on the rates and amplitudes of the fast and slow phases (Fig. 3, A and C, and Table III). A large deuterium isotope effect has previously been observed for NADPH reduction of the S457A mutant (18). Similarly, large deuterium isotope effects were also observed for the Asp675 and Cys630 mutants; these were associated with the phase having the larger A452 change, suggesting that hydride transfer occurred in this phase.


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Fig. 3.   Kinetics of anaerobic reduction of mutant P450R proteins by NADPH. Protein solutions were made anaerobic as described under "Materials and Methods," and NADPH was added to give a 5:1 ratio of NADPH to protein. A452 values are normalized to 1 for comparison purposes. Lines are fits of data points to Equation 6 or 7. A and B show A452 and A585 changes for wild-type (), D675N (open circle ), and C630A (black-triangle) proteins. C and D show A452 and A585 changes for D675V (), S457A/D675N (open circle ), and S457A (black-triangle). E and F show A452 and A585 changes for S457A/D675N/C630A.

                              
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Table III
Reduction 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 rate constants k1 and k2 for reduction. Data were obtained from at least two separate protein preparations and are expressed as mean ± S.D. (n). The wild-type protein was reduced under aerobic conditions. The mutant proteins were reduced under anaerobic conditions.

Reduction of the D675N mutant by NADPH under anaerobic conditions resembled that of wild-type enzyme, with 84% of the total absorbance change and the isotope effect occurring in the first phase. Rate constants were decreased 50- and more than 500-fold, respectively, for the first and second phases of reduction (Fig. 3A and Table III). In contrast, NADPH reduction of four other mutants (D675V, S457A/D675N, C630A, and S457A/D675N/C630A) differed in that most of the A452 change and the isotope 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 associated with the second phase of reduction (Fig. 3C and Table III), as was the isotope effect. Replacement of Cys630 with alanine produced approximately 20-fold decreases in both k1 and k2, with 80% of total absorbance change associated with k2 (Fig. 3A and Table III). The isotope effect was also associated primarily with k2. Substitution of all three residues resulted in an extremely slow rate of reduction by NADPH, characterized by a transient initial phase, followed by a second phase which exhibited the isotope effect and 93% of the total A452 change (Fig. 3E and Table III). k2 for this second phase was 104-fold lower than wild-type k2 and 5 orders of magnitude lower than wild-type k1.

Formation of wild-type flavin semiquinone as monitored by absorbance changes at 585 nm was concomitant with flavin reduction, suggesting rapid interflavin electron transfer (34-38). S457A semiquinone formation was biphasic, with an initial rate comparable with that of flavin reduction (Fig. 3D and Table III; Ref. 18). A585 changes for the D675N and S457/D675N mutants, however, were small and slower than the corresponding rates of flavin reduction (Fig. 3, B and D, Table III). The A585 changes observed for the D675V, C630A, and S457A/D675N/C630A mutants were biphasic, with a transient first phase followed by a second, slower phase with rate constants comparable with the second, slow phase of flavin reduction (Fig. 3, B and F; Table III).

Anaerobic NADPH Titration of P450R-- Anaerobic NADPH titration was used to investigate the effects of the Asp675 and Cys630 substitutions on flavin redox potential. Titration of rat liver P450R with NADPH under anaerobic conditions has been shown to produce visible absorption spectra that are characteristic for each redox state of the enzyme (31, 39). The addition of 0.5 mol of NADPH/mol of enzyme to the fully oxidized protein produces the spectrum of the air-stable semiquinone (FAD/FMNH·), characterized by an absorbance decrease at 452 nm, isosbestic points at 363 and 502 nm, and the appearance of a long wavelength absorbance with a maximum at 585 nm and a shoulder at 630 nm. The further addition of NADPH produces the 3e--reduced form (FMNH2/FADH·), with a further absorbance decrease at 452 nm, minimal absorbance changes at 585 nm, and a slightly altered long wavelength absorbance (lambda max at 593 nm and shoulder at 630 nm) characteristic of the FAD semiquinone (FADH·). Since the midpoint potential of the FADH·/FADH2 couple is below that of NADPH, NADPH is unable to reduce P450R to the 4e--reduced stage (31, 39) and the further addition of NADPH to the 3e--reduced enzyme produces minimal absorbance decreases at 452 nm. Identical absorbance changes are seen upon NADPH titration of the recombinant enzyme, while the S457A titration produces spectra consistent with a decrease in the FAD/FADH· redox potential (18).

Fig. 4 presents the absorbance changes at 452 and 585 nm observed upon the addition of NADPH under anaerobic conditions to the wild-type and mutant proteins. Reduction of the D675N protein to the 1e--reduced (FAD/FMNH·) stage paralleled that seen for the wild-type enzyme (Fig. 4, A and C), with production of the air-stable semiquinone upon the addition of approximately 0.5 mol of NADPH/mol of protein. However, the further addition of NADPH to the D675N protein produced minimal changes in A452 but a decrease in A585, consistent with reduction of FMNH· to FMNH2 and no formation of FADH·. This behavior is similar to that reported previously for the S457A mutant and suggests that the D675N mutation, like the S457A mutation, decreases the FAD/FADH· redox potential. In contrast, the S457A/D675N (Fig. 4D) double mutant required nearly 2 mol of NADPH/mol of protein for full production of the 1e--reduced protein, with no further absorbance changes at either wavelength observed upon further addition of NADPH, consistent with a decreased FAD/FADH2 redox potential.


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

Reduction of the D675V mutant to the 1e--reduced stage required approximately twice as much NADPH as the wild-type protein (Fig. 4B) and the further addition of NADPH produced no further decreases in A452 and a slight decrease in A585, consistent with a decreased FADH·/FADH2 redox potential. NADPH reduction of the C630A mutant proceeded in a manner similar to wild type (Fig. 4E), although the magnitude of the decrease in A452 was slightly smaller than that of wild type. Finally, formation of the 1e--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 further addition of NADPH, suggesting that both the FAD/FADH· and FAD/FADH2 redox potentials were decreased (Fig. 4F).

    DISCUSSION

We have previously shown that Ser457 is a residue crucial for hydride transfer and control of FAD redox potential (18). The present studies demonstrate that Cys630 and Asp675 of P450R are also located in the active site for FAD reduction and have a key 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 and have minimal effects on NADPH and NADP+ binding and flavin content. The decrease in Kmcyt c concomitant with the decrease in kcat is consistent with the nonclassical two-site ping-pong mechanism proposed for P450R (32). For each of the active site mutants examined, the decreases in kcat parallel changes in the rate of flavin reduction and are associated with large NADPD isotope effects, indicating that the effects of these substitutions on catalysis are primarily a result of impaired hydride transfer. Activities of multiply-substituted reductase mutants were roughly multiplicative, strongly suggesting that these residues interact to facilitate hydride transfer.

Comparison of the effects of the D675V and D675N substitutions suggests that hydrogen bond formation between Asp675 and some other group, probably Ser457, is necessary for hydride transfer. kcat for the D675N mutation, which retains hydrogen bonding capacity, is decreased only 14-fold, while elimination of hydrogen bonding interactions with the D675V and S457A/D675N mutants decreases kcat 700-fold. The ionic strength dependence of cytochrome c reduction is also shifted to higher ionic strengths by the D675N mutation (data not shown), consistent with the presence of a stronger hydrogen bond with this mutant. Although Ser457 and Asp675 do interact to influence hydride transfer, the effect of removing the Ser457 hydroxyl group (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, which could partially restore activity. Alternatively, the D675V substitution could interfere with some other step in addition to hydride transfer, such as formation of the enzyme-NADPH complex or NADP+ release. X-ray crystallography of FNR indicates 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 the N-5 of FAD (20). The P450R crystal structure indicates that Ser457 is within hydrogen bonding distance of the flavin N-5, but hydrogen bonding between Ser457 and Asp675 has not been established (15).

Previous results have demonstrated dependence of wild-type kcat on a macroscopic pKa of approximately 6.9 (21). This macroscopic pKa could reflect deprotonation of a single functional group, which in turn could be influenced by other ionizable groups in the microenvironment of the protein. Elimination or perturbation of this pKa by mutagenesis provides a means of investigating the group(s) involved. Results from this study show that, although Asp675 is necessary for catalysis, its pKa does not fall between 5.5 and 10, and some other group is responsible for the 6.9 pKa. This unknown acidic group is influenced by Cys630; removal of the sulfhydryl increases the pKa by nearly 1 pH unit. Since catalytic activity is virtually abolished by the S457A/D675N/C630A triple mutation, it is unlikely that there is another, unidentified catalytic amino acid. 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 proton from Cys630 could stabilize the reduced FADH2, thereby increasing the redox potential and facilitating hydride transfer; this is consistent with the decreases in FAD redox potential and rate of hydride transfer observed with the C630A mutant.

The Asp675 mutations as well as the C630A, S457A, and other carboxyl-terminal mutations2 all shift the pKa of the basic group from 8.8 to >10. This may be due to a shift in the rate-limiting step from this pH-dependent step to the hydride transfer step.

Reduction of P450R by NADPH proceeds through rapid formation of one or more charge-transfer complexes (35, 41), followed by slower hydride transfer to FAD; hydride transfer is largely rate-limiting for cytochrome c reduction (32). Biphasic absorbance changes at 452 and 585 nm are observed upon NADPH reduction of wild-type reductase. The first phase occurs at a rate consistent with enzyme turnover and corresponds to FAD reduction and rapid interflavin electron transfer, while the second, slow phase has been proposed to correspond to reduction of the 2-electron-reduced reductase by a second molecule of NADPH and is limited by the rate of NADP+ release and/or some conformational change (35). Consistent with this hypothesis, a deuterium isotope effect for reduction of the wild-type enzyme by NADPD is observed only for the first phase but not the second (18).

Like the wild-type enzyme, reduction of the D675N protein by NADPH produces biphasic kinetics. The rate of the first phase, k1, is consistent with kcat (Table III), while the rate for the second phase is slower than that of overall turnover and shows no deuterium isotope effect. In contrast, although NADPH 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. This suggests that the second phase, with rate constant k2, represents hydride transfer, and k1 may represent some prior step, perhaps the formation of a P450R-NADPH pre-hydride transfer complex. Pre- and post-hydride transfer complexes have been observed spectroscopically for other members of this family, such as FNR (42), phthalate dioxygenase reductase (43), and P450-BM3 (44), and postulated, but never observed, in the case of P450R (35, 41). The greatly decreased rate of hydride transfer and, perhaps, alterations in the conformation of the NADPH-FAD complex allow observation of the pre-hydride transfer complex in these proteins.

Results of the anaerobic NADPH titrations indicate that these substitutions modulate FAD redox potential (Fig. 4; Ref. 18). The D675N and S457A/D675N/C630A mutants, like the S457A mutant, destabilize the FADH· semiquinone, while the D675V and S457A/D675N (and possibly the Cys630) mutations decrease the FADH·/FADH2 redox potential.

While rates of semiquinone formation are roughly compatible 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 accounted for), semiquinone formation for the D675N and S457A/D675N mutants is slower than kcat. A similar phenomenon occurs with FNR; recently, Medina et al. (45) have demonstrated destabilization of the FADH semiquinone and inhibition of activities requiring 1e- transfer in the Glu301 mutant of FNR, which is homologous to Asp675.

A model for hydride transfer consistent with the kinetic data is presented in Fig. 5. Ser457 stabilizes the reduced flavin by accepting a hydrogen bond from the reduced N-5. Asp675 hydrogen bonds to Ser457, orienting Ser457 and/or making it a stronger hydrogen bond acceptor. These residues may also act to position the flavin and nicotinamide in an orientation suitable for hydride transfer. Cys272 of FNR, which is equivalent to Cys630 of P450R, is located about 6 Å from the flavin N-5, and modeling studies propose that it may contact the nicotinamide ring (12). We propose that Cys630 interacts with both the FAD and the nicotinamide and may catalyze hydride transfer by destabilizing the nicotinamide C-4-H bond and/or donating a proton to the reduced flavin. In this position, it may also be able to accept a proton from the anionic FAD semiquinone (FADH-) after electron transfer to FMN to yield the observed blue neutral semiquinone.


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Fig. 5.   Model for hydride transfer in P450R. The arrow indicates the direction of hydride transfer. Heavy dotted lines indicate hydrogen bonding interactions of Ser457. The thin dotted lines indicate proposed hydrogen bonds involving Cys630.

Although the residues corresponding to Ser457, Cys630, and Asp675 are conserved in all members of the FNR family and mutagenesis of corresponding residues of FNR, Ser96 and Cys272, decreases FNR catalytic activity and FAD redox potential (46, 47), the active sites of these proteins are designed for the different functions carried out by these enzymes: NADPH oxidation coupled to substrate reduction in P450R, nitric-oxide synthase, 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 the relative redox potentials and the orientation of the flavin and pyridine rings in some optimum geometry. The FAD redox potential is dependent upon the stabilization of the anionic reduced flavin, which is influenced by hydrogen bonding interactions between the protein and N-1, O-2, O-4, and N-5 of the flavin. The large isotope effects obtained with these mutants are suggestive of the presence of tunneling in the hydride transfer, which requires a precise geometry as well as a short distance between the flavin N-5 and nicotinamide C-4. It appears that the relative positions of these residues are critical in determining both the rate and equilibrium of the hydride transfer reaction; crystallographic analysis of these mutants may identify the hydrogen bonding patterns important for hydride transfer and control of flavin redox potential.

    ACKNOWLEDGEMENTS

We are grateful to Timothy Culligan and John Sheehan for technical assistance with protein purification, to Dr. Brian Fox and Dr. Dexter Northrop for use of the stopped-flow spectrophotometers and for helpful discussions, and to Mary Jo Markham and Kristen Adler for preparation of this manuscript.

    FOOTNOTES

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

Dagger To whom correspondence should be addressed.

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

    ABBREVIATIONS

The abbreviations used are: P450R, NADPH-cytochrome P450 oxidoreductase; FNR, ferredoxin-NADP+ reductase; PCR, polymerase chain reaction; AcPyrADP, 3-acetylpyridine adenine dinucleotide phosphate; e-, electron equivalent(s); CAPS, 3-(cyclohexylamino)propanesulfonic acid.

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Abstract
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