Journal of Inorganic Biochemistry 97 (2003) 265–275www.elsevier.com/ locate/ jinorgbio

E xamining the mechanism of stimulation of cytochrome P450 bycytochrome b : the effect of cytochrome b on the interaction between5 5

cytochrome P450 2B4 and P450 reductasea , b*James R. Reed , Paul F. Hollenberg

aDepartment of Drug Metabolism, Merck and Co., PO Box 2000, Rahway, NJ 07065,USAbDepartment of Pharmacology, University of Michigan, Medical Science Research Building III, 1150 West Medical Center Drive, Ann Arbor,

MI 48109-0632,USA

Received 10 April 2003; received in revised form 19 June 2003; accepted 28 June 2003

Abstract

Dissociation constantsK for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence ofd

various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b (b )5 5

stimulates metabolism of this substrate and dramatically decreases the substrate-relatedK values. Experiments are performed to test if thed

b -mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b (Mn b ) on5 5 5

product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b stock solution that have been5

shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate ofproduct formation. Thus, the electron transfer capability of b is necessary for stimulation of metabolism. Furthermore, stopped flow5

spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P4502B4-mediated metabolism improves, in the presence of Mn b , with slower delivery of the first electron of the catalytic cycle by the5

reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of theP450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced,but not oxidized, reductase is bound to the P450. 2003 Elsevier Inc. All rights reserved.

Keywords: Cytochrome P450; Cytochrome b ; P450 reductase; Conformational dynamics5

1 . Introduction electron in the P450 catalytic cycle[3,8,9]. The latter isthought to be the rate-limiting step for many P450-me-

Cytochrome b (b ) is a ubiquitous heme protein with a diated monooxygenase reactions[10].5 5

mass of 17 kDa that has a variety of cellular functions It is also thought that the interaction of P450 with b5

[1–3]. It has diverse effects on cytochrome P450 (P450) may induce a conformational change in the P450 whichreactions. Depending on the P450 and the substrate facilitates catalysis. The most compelling evidence for thismetabolized, b has been reported to stimulate[4–6], effect comes from studies in which apo b (which is5 5

inhibit [4,5], or have no effect[4,6] on P450-mediated incapable of participating in redox reactions with P450)metabolism. In some cases, the presence of cytochrome b was shown to activate several P450-mediated reactions5

is an obligate requirement for the functional reconstitution [11,12]. However, these findings are somewhat contro-of P450-mediated activity[7]. The mechanism by which b versial because other investigators have not seen stimula-5

influences P450 reactions remains controversial. There is tion by apo b when studying some of the same reactions5

considerable evidence to suggest that b stimulates P450[13]. Furthermore, recent data suggest that some of the5

catalysis by increasing the rate of delivery of the second stimulatory effects of apo b in these reactions may be5

attributed to the scavenging of heme from partially dena-tured cytochrome P450[14].*Corresponding author. Tel.:11-732-594-6014; fax:11-732-594-

The effect that b -induced conformational changes may1416. 5

E-mail address: james reed@merck.com(J.R. Reed). have on cytochrome P450 activity is even more unclear in]

0162-0134/03/$ – see front matter 2003 Elsevier Inc. All rights reserved.doi:10.1016/S0162-0134(03)00275-7

266 J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275

light of studies which reported no stimulation (and, in fact, ing the rate of association between the proteins. Theeven inhibition) of P450 reactions when a redox-inactive former effect could stimulate the P450-mediated reactionderivative of native b made by reconstituting apo b with by allowing both electrons to be delivered in a single5 5

a Mn protoporphyrin IX prosthetic group (Mn b ) was binding event, whereas the latter effect could shorten the5

substituted for the heme containing protein[6]. Thus, the delay between the delivery of the first and second elec-evidence that b can induce stimulatory conformational trons.5

changes in P450 remains dubious. It is generally assumed that the presence of substrateOn the other hand, stimulation of P450 reactions by invariably lowers the dissociation constant,K , for thed

b -induced changes in conformation might be inferred binding of P450 reductase and P450[23,24]. However, a5

from the observation that the binding of b to P450 results study of the effect of substrate on both the spin state and5

in an increase in the amount of high spin P450[15,16]. the rate of first electron reduction of P450 2B4[25]This alteration in spin state is thought to indicate a change showed that, in contrast to most substrates, while amino-in the conformation of P450[17], and has been associated pyrine increased the amount of high spin P450 (type Iwith the catalytic activity of P450 2B enzymes[9,18]. binding), it lowered the rate of association between P450Furthermore, the conformational changes associated with and P450 reductase. In order to confirm that the bindingthis increase in high spin P450 also typically lower theK between reductase and P450 is reduced in the presence ofd

of substrate binding to P450[16,19]. Thus, changes in aminopyrine, we have measured the effect of this substrateP450 spin state associated with a b -induced conformation on theK of P450 and reductase. We also compared the5 d

change might be expected to result in increased P450 effects of a variety of other substrates in order to determineenzymatic activity. However, a more direct mechanism for if the effect of aminopyrine on the interaction of the twostimulation of P450 catalytic activity by the interaction proteins is unique. As shown herein, our results support thewith b is discussed below and is tested in the current original finding of Backes and Eyer[25].5

study. We thought that the potential for conformation-mediatedPrevious work has shown that the oxyferrous form of stimulation of P450 by b (via the mechanism described5

P450 can either be reduced by ferrous b or oxidized by above) would be greatest with a substrate, such as amino-5

ferric b [20]. From studies using a cross-linked P450 2B4 pyrine, which greatly reduces the binding affinity between5

and b derivative that retained enzymatic activity, a model P450 and reductase. Thus, in this study, we also measured5

was proposed to explain the mechanism by which cyto- the effect of b on the P450-mediated metabolism of5

chrome b stimulates the rate of second electron transfer aminopyrine and theK for reductase and P450 in the5 d

[21]. In this model, cytochrome b rapidly oxidizes the presence of the substrate. A b -mediated reduction inK5 5 d

ferrous P450 formed after one electron reduction by P450 and increase in the rate of metabolism of aminopyrinereductase. This results in the formation of a quaternary would be consistent with a mode of stimulation involvingcomplex comprised of ferric P450:P450 reductase:sub- conformational dynamics. In order to verify that thesestrate:ferrous b . The ferric P450 is then reduced a second trends were attributable to this mechanism, we also tested5

time by the P450 reductase to allow for the formation of the redox-inactive Mn b on metabolism of aminopyrine.5

the oxyferrous P450 species and continuation of the The Mn b appears to bind to P450 in a manner similar to5

catalytic cycle with the delivery of the second electron that of the native protein[26]. Thus, if stimulation occursfrom the ferrous b . The net result is that both electrons through a b -mediated conformation change, Mn b also5 5 5

required for P450-mediated catalysis are delivered in a should activate the reaction.single binding event with the P450 reductase. The rate of Cytochrome b -related stimulation of P450 reactions is5

catalysis is presumed to be increased by obviating the believed to involve improvements in reaction couplingnecessity for a second P450 and reductase-binding event[27,28] Therefore, the reaction stoichiometry for P450,

that might be required for delivery of the second electron if 2B4-mediated metabolism of various substrates was de-dissociation of the reductase and P450 occurred before termined to see if the effects of Mn b could be related to5

both electrons could be transferred to the P450. coupling. In this case, we hypothesized that Mn b would5

This model explains the manner by which the redox- dramatically improve the coupling of reactions with sub-related activity of b could function to stimulate P450- strates that reduce the tendency for P450 and P4505

mediated metabolism. However, it also accommodates a reductase to interact.mechanism for b -related stimulation that involves an5

effect on the conformation of the P450 protein. Morespecifically, the binding of b may alter the conformation 2 . Materials and methods5

of the P450 in such a way that the binding affinity betweenthe P450 and P450 reductase is enhanced[22]. Such an 2 .1. Materialseffect could ultimately result in the stimulation of P450-mediated catalysis by either lengthening the time of Benzphetamine, aminopyrine, resorufin,L-a-dilauryl-sn-interaction between the reductase and P450 or by increas- glycero-3-phosphocholine (DLPC), hemin, and NADPH

J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275 267

21 21were purchased from Sigma (St. Louis, MO). Benzylox- sorbance coefficient (6.22 mM cm ). The readingsyresorufin (BRF), pentoxyresorufin (PRF), 7-ethoxy-4-tri- were recorded on a Shimadzu UV-2501 PC UV–Visfluoromethylcoumarin (7EFC), and 7-hydroxy-4-trifluoro- recording spectrophotometer with a Shimadzu TCC-con-methylcoumarin (7HFC) were purchased from Molecular troller. The background rate of the P450 reductase-cata-Probes (Eugene, OR). Formaldehyde (37%), hydrogen lyzed oxidation of NADPH was determined by reconstitut-peroxide (30%), and solvents of the highest purity avail- ing P450 reductase without P450 and was subtracted fromable were purchased from Fisher Scientific (Pittsburgh, the rate determined in the presence of the P450. ThisPA). Manganese protoporphyrin IX was purchased from background rate of oxidation was also determined withPorphyrin Products (Logan, UT). each of the different substrates tested. Only the two

alkoxyresorufin substrates affected the rate of NADPH2 .2. Enzyme sources consumption by P450 reductase as they have been shown

to participate in a reductase-catalyzed redox cycle withCytochrome b was purified from the livers of male molecular oxygen and superoxide[35]. The ratio of the5

Long Evans rats (175–190 g) according to the procedures measured rate of NADPH oxidation to the maximal rateof Waxman and Walsh[29]. Cytochrome b was induced was taken to represent the fractions of reductase and P4505

by treating rats with phenobarbital (0.1% in the drinking that were bound to one another[15,23]. Thus, a Scatchardwater for 12 days). The apo b was prepared by precipi- plot could be generated from the data by treating the5

tation of the native b with acid acetone as described[11]. reductase as the ligand and the P450 as the receptor as5

The method of Morgan and Coon[6] was used to described by Tamburini and Gibson[15]. This plot wasreconstitute the apo b with manganese protoporphyrin IX then used to determine theK .5 d

to form the Mn b . A UV–Vis spectrum of the apoprotein5

showed that it contained less than 0.2% of the native b 2 .4. Enzyme assays5

(data not shown). P450 2B4 was purified as described[30]from the livers of New Zealand white male rabbits which The rates of the reactions for theO-dealkylation ofwere given 0.1% phenobarbital in drinking water for 7 7EFC and theN-demethylation of aminopyrine weredays. Most experiments used rat P450 reductase which was determined as described by Hanna et al.[36]. Unlessexpressed and purified fromE. coli as described[31]. specified otherwise, the reaction mixtures contained 0.05

The concentrations of the cytochromes P450 were or 0.0125 M potassium phosphate buffer (pH 7.4) (thedetermined by the method of Omura and Sato[32]. The former was used for the 7EFC assay and the latter forconcentrations of b were determined spectrally using the aminopyrine), 0.2mM P450 2B4, 40mg/ml bovine serum5

21 21extinction coefficient of 117 mM cm at 413 nm for albumin, 400mg of DLPC/nmol of P450 enzyme, andthe ferric form [33]. The concentration of apo b was equimolar amounts of P450, reductase, and cytochrome b5 5

determined by titration with a basic heme solution in 50% (when added). Unless specified otherwise, the stock solu-ethanol as described[26]. The concentration of Mn b was tions containing b , apo b , and Mn b were 2.2mM and5 5 5 5

determined spectrally using an extinction coefficient at 469 contained 20 mM Tris (pH 8.1), 0.2 mM EDTA, and 10%21 21nm of 57 mM cm [26]. The concentration of P450 glycerol. The reaction volume was 1 ml. The reactions, at

reductase was determined from the absorbance of the 308C, were initiated with 200mM (final concentration)oxidized enzyme at 456 nm using an extinction coefficient NADPH.

21 21of 21.4 mM cm [34].2 .5. Determination of reaction stoichiometry

2 .3. Determination of the K values for binding of P450d

and reductase For these experiments, the proteins were reconstituted asdescribed above. Water and buffers were run through

The dissociation constant for reductase and P450 bind- chelex columns to remove trace metals that might interfereing was determined by measuring the rate of NADPH with hydrogen peroxide determinations by initiating freeoxidation at different P450 reductase:P450 ratios as de- radical reactions. For the reconstitutions, P450 and P450scribed by French et al.[23]. The P450 enzyme (0.5mM, reductase were incubated together at equimolar concen-final concentration) was reconstituted with five different trations on ice for 4 min before adding the sonicated DLPCconcentrations of P450 reductase (0.1, 0.2, 0.4, 0.5, or 2 (from a 1 mM aqueous solution). This mixture then sat onmM) in 50 mg/ml DLPC in the presence or absence of the ice for at least 40 min before it was suspended insubstrates indicated. The reductase preparations used for potassium phosphate assay buffer. The final volume of thethese determinations contained no degraded form of the reactions was 1 ml. The reactions used 200 pmol of P450enzyme (as determined electrophoretically) and were and 0.13 mM (final concentration) DLPC. High concen-frozen only one time at280 8C. The rate of NADPH trations ($0.2mM) of Mn b were required in all reactions5

oxidation was determined at 308C by measuring the loss to avoid having a significant portion of the proteinof absorbance at 340 nm with time and using the ab- disassociating to Mn protoporphyrin IX and apo b[26].5

268 J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275

Depending on the experiment, two different methods were rate of delivery of the first electron of the catalytic cycle,used to add cytochrome b . If the objective was to study was determined essentially as described previously[41,42].5

the effects associated with b -related (or Mn b -related) Equimolar amounts of P450 2B4 and P450 reductase (25 5

inhibition of the rate of NADPH oxidation by P450, the b mM, final concentration) were reconstituted in 250mg of5

was added from a 2.2mM stock immediately after DLPC as described above. After a minimum of 40 min, thesuspending the protein and lipid mixture in phosphate proteins were taken up in 0.05 M potassium phosphatebuffer (i.e. after the 40-min pre-incubation period de- buffer (pH 7.4). If b or Mn b were present, they were5 5

scribed above). Substrate was then added at the con- then added at a final concentration of 2mM. The stockcentrations indicated, and the samples were immediately solution of b was 32mM, whereas that of Mn b was 145 5

incubated for 5–10 min at 308C in the Shimadzu instru- mM. The protein sample was placed in a tonometer, and 5ment described above. The reactions were then initiatedml of PCD (0.75 U) and 200mM PCA (which was placedwith 200mM (final concentration) NADPH. This protocol in the side arm of the tonometer) were added as an oxygenwas found to maximize the inhibitory effect of b . scavenging system[10]. The solution containing PCD was5

Alternatively, if the objective was to examine the effect of overlaid with argon containing#0.5 ppm O . After ten2

b on reaction stoichiometry when the latter did not inhibit cycles of alternating gas and vacuum exchange, the sample5

NADPH oxidation, the b stock solution was diluted to 0.2 was overlaid with CO, and PCA was then added from the5

mM before adding the protein to the reaction mixture. The side arm of the tonometer. A syringe filled with a solutionrest of the protocol was the same as that described above. containing 0.4 mM NADPH was bubbled with argon for

The oxidation of NADPH was monitored at 340 nm as 10 min. Care was taken to ensure that both solutions haddescribed above. After 4 min, the sample was transferred equal concentrations of glycerol. Equal volumes of bothto an Eppendorf tube. At 4.25 min, 0.3 ml of sample was solutions were mixed in a Hi-Tech Scientific Model SF-61transferred to another tube containing 0.5 ml of ice-cold stopped flow spectrophotometer controlled by a Mac IICXtrichloroacetic acid solution (3%). This mixture was using KISS software (Kinetic Instruments). Reduction ofvortexed and then used to measure H O production by the P450 was followed at 308C for 4 min. In the absence of b2 2 5

ferrithiocyanate method[37]. At 4.5 min, the remaining or Mn b , the absorbance changes with time were moni-5

0.7 ml of sample was mixed with either 238ml of ice-cold tored at 450 nm. In the presence of the two proteins,acetonitrile or 77ml of a solution comprised of 25% changes at the respective isosbestic points were measuredZnSO :500 mM semicarbazide (pH 7–8) (10:1). The (440 nm for b and 454 nm for Mn b ).4 5 5

former was used to quantitate the 7-hydroxy-4-trifluoro- The kinetic data were analyzed using a parallel exponen-methylcoumarin (7HFC) formed from the 7EFC reaction, tial fitting routine based on the Marquardt-Levenbergwhereas the latter was used in the quantitation of formalde- algorithm with four irreversible exponential steps[43].hyde in the benzphetamine and aminopyrine reactions. An Initially, rate constants and amplitudes were estimated byadditional 70-ml aliquot of saturated Ba(OH) solution was visual inspection and were fixed in the modeling program2

added for the formaldehyde determination, and the samples to give a first approximation of a fit to the data. Thewere vortexed again after adding the ZnSO :semicarbazide program was then allowed to optimize the fit by varying4

solution. The rate of excess water formation was calculated the kinetic constants in an iterative process. Conditionsas the difference between the rate of NADPH oxidation were repeated in triplicate, and the optimized rate constantsand the sum of the rates of product and hydrogen peroxide were fixed when performing the fitting routine with theformation divided by 2 because four electrons, or two repeat measurements in order to derive error values for theNADPH molecules, are presumed to be consumed in the amplitudes of the phases.production of excess water[38].

It has been reported that a minor metabolite is formed inaddition to 7HFC during the metabolism of 7EFC by rat 3 . Resultsliver microsomes[39], whereas the metabolism of amino-pyrine by rabbit and rat liver microsomes has been shown 3 .1. Effect of substrate on the K values for binding ofd

to produce only the demethylated product, 4-aminoan- P450 and reductasetipyrine [40]. In a previous study[9], the contribution ofthis side reaction of 7EFC metabolism did not significantly Table 1shows the effect of various compounds on theaffect the stoichiometry of the metabolism by P450 2B4 K values for binding of P450 reductase to P450. Asd

with and without b . shown in the table, aminopyrine lowers the affinity be-5

tween the reductase and the P450 as it significantly2 .6. Anaerobic reduction of P450 2B4 by P450 increases theK . As may be expected from the previousd

reductase study[22], the addition of b results in a dramatic decrease5

in theK for binding of the two proteins. Our findings alsod

The effect of b and Mn b on the anaerobic reduction suggest that BRF slightly increases theK for P4505 5 d

of 2B4 by P450 reductase, which presumably reflects the reductase binding to P450. However, the determinations

J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275 269

T able 1 measurements may not be trueK values. As a result, wedaDissociation constants for the binding of P450 reductase to P450 2B4 also determined theK for reductase and P450 in thed

Substrate P450 2B4/rat absence of substrate and in the presence of 7EFC byreductase (nM) measuring the type I spectral change of P450 2B4 in the

bNone 123, 114 presence of reductase[15,23]. The resulting measurementsAminopyrine (2.5 mM) 417 agree very well with the determinations based on theAminopyrine1b 395 steady state NADPH oxidation rate (Table 1). With P450BRF (5 mM) 164

b 2B4, we previously showed that cytochrome b stimulated57EFC (100mM) 81, 89the metabolism of all of the substrates except benzpheta-Benzphetamine (375mM) 41

PRF (5mM) 110 mine [9], so there is no correlation between the effects ofErythromycin (500mM) 53 b on catalysis and the tendency to reduce the affinity5

a between P450 2B4 and P450 reductase.The dissociation constants (K ) for the interactions of P450 with P450d

reductase in the presence of different substrates were determined bymeasuring the rates of NADPH oxidation at different P450 reductase 3 .2. Effects of Mn b and b on P450 2B4-mediated5 5concentrations as described in Materials and methods. The numbersmetabolismrepresent values derived from the average of duplicate measurementstaken at each reductase concentration. In all cases, the two determinations

The effects of Mn b on the metabolism of eithernever varied by more than 20% from one another. When the effects of b 55

were tested, the protein was added from a concentrated stock solution (32aminopyrine or 7EFC by P450 2B4 were determined frommM) immediately before the pre-incubation at 308C (see Materials and the stoichiometries of the respective reactions (Table 2).methods). The maximal rate of NADPH oxidation by P450 2B4 in the For reasons which are explained below, the Mn b was5metabolism of aminopyrine was inhibited by 30% by the b in these5 tested under a variety of reconstitution conditions. Underdeterminations. All other rates were increased upon addition of substrate

identical conditions to those used with b in the table (Mn(data not shown). 5b The K value was measured by titrating 2mM P450 2B4 with b (concentrated)), the Mn b had no effect on the productd 5 5

reductase and measuring the magnitude of the type I spectral changeformed from aminopyrine and inhibited that from 7EFC.[15,23]. Conversely, both reactions were activated by b . Under the5

conditions tested, both b and Mn b inhibited the rate of5 5

with the alkoxyresorufins are complicated by the fact that NADPH oxidation for both substrates whereas b only5

the P450 reductase reduces these substrates to forms that inhibited NADPH oxidation when 7EFC was the substrate.cannot be metabolized by the P450[44]. As shown in the In all cases, Mn b inhibited the rate significantly more5

table, all of the other compounds reduced theK for than b . Consistent with previous findings[27,28], cyto-d 5

reductase binding to the respective P450 enzymes. Because chrome b dramatically improved reaction coupling (de-5

it could be argued that the rate of NADPH oxidation is fined as the percentage of NADPH oxidation utilized in therepresentative of a multi-step process and not just the formation of product) when stimulating the respectivecomplex formation between reductase and P450, our reactions. Surprisingly, Mn b also improved the coupling5

T able 2aReaction stoichiometry for metabolism by P450 2B4: the effects of Mn b5

Aminopyrine NADPH Desmethyl- H O Excess H O2 2 2

(2.5 mM) oxidation aminopyrine

Control 1 10.8360.4 1.6060.4 (15) 7.160.7 (65) 1 (20)1b (conc.) 11.0261.2 2.9560.1 (27) 6.061.5 (54) 1 (19)5

Control 2 13.7861.8 2.2660.2 (16) 10.761.4 (78) 1 (6)1Mn b (dilute) 14.3461.6 2.2160.4 (15) 8.360.4 (58) 2 (27)5

1Mn b (conc.) 9.2061.2 2.2660.3 (25) 2.460.6 (27) 2 (49)5

123Mn b (conc.) 8.1461.3 1.7260.2 (21) 1.561.2 (19) 3 (60)5

7EFC NADPH 7HFC H O Excess H O2 2 2

(100 mM) oxidation

Control 40.4063.8 3.5260.1 (8.7) 42.266.6 (100) –1Mn b (conc.) 21.5060.6 2.4660.1 (12) 13.464.4 (62) 3 (26)5

1b (conc.) 31.2663.4 8.8760.4 (28) 11.064.8 (35) 6 (37)5

a The stoichiometries of the P450 2B4 reactions were determined as described in Materials and methods. The numbers represent the averages6S.D. oftriplicate determinations. The units of the reactions are in nmol product /min per nmol of P450. The numbers in parentheses represent the percentage ofNADPH oxidation that is utilized in the formation of the different products. The substrate tested and its concentration is listed at the top of each subheadingin the table. The control reaction mixtures contained substrate and equimolar concentrations of P450 reductase and P450 2B4 reconstituted in DLPC. Adifferent control was used for the experiments testing b and Mn b with aminopyrine. Either b or Mn b were added at concentrations equimolar to the5 5 5 5

P450.

270 J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275

of both reactions even though neither was stimulated to intermediate would be strong evidence that b could5

form increased amounts of product. The amount of excess influence metabolism by effecting changes in P450 con-water was increased upon addition of Mn b . formation. In order to address this possibility initially, we5

determined the effects of Mn b and b on the first electron5 5

3 .3. The effects of b and Mn b on the rate of reduction of P450 2B4 by P450 reductase (Table 3).5 5

anaerobic reduction of P450 2B4 by P450 reductase Because the effects of Mn b and b on the rates of5 5

NADPH oxidation and hydrogen peroxide formation wereThe stoichiometry data for the metabolism by P450 2B4 more dramatic with 7EFC, we used this substrate to

(Table 2) indicate that Mn b improves the coupling of the compare the rates of first electron reduction in the presence5

reactions with both substrates. Despite the improved and absence of b and Mn b . If the rate of reduction is not5 5

coupling, which can be attributed to a reduction in the rate inhibited by b or Mn b , the lower rate of hydrogen5 5

of hydrogen peroxide formation, Mn b had no effect on peroxide in the presence of Mn b would have to indicate5 5

the rate of product formation. The formation of hydrogen increased stabilization of the oxyferrous intermediateperoxide in P450 2B4-mediated metabolism has been because the hydrogen peroxide originates from the releaseshown to result from dismutation of the superoxide. This of superoxide[28,45]. In the reduction experiments, b and5

by-product forms from both reductase-mediated oxygen Mn b were added from concentrated stock solutions (.25

reduction and decay of the oxyferrous intermediate in the mM). In Table 3,phase 1 is attributed to the reduction ofP450 catalytic cycle[28,45]. Because of the potential P450 reductase by NADPH and is characterized by aeffect of the P450 on the reductase-mediated process, it is decrease in absorbance at the observed wavelengths. Thenot possible to determine the relative contribution by the P450 reduction was modeled with three phases (phasestwo pathways[28]. However, one possible interpretation of 2–4). As shown in the accompanying manuscript[67], atthe stoichiometry data is that the Mn b is stabilizing the least one of these phases can be eliminated when the ratio5

oxyferrous intermediate and, in turn, preventing the release of reductase:P450 is changed from 1:1 to 3:1. Thus, asof superoxide. In doing this, the Mn b may indirectly shown previously by Backes and Eyer[25], the reduction5

decrease the rate of futile oxidation of NADPH. of P450 2B4 by reductase can be modeled as a biphasicInhibition of the P450-mediated steady state rate of process.

NADPH oxidation upon addition of b has been reported The effect of b and Mn b on the reduction of P4505 5 5

previously and the magnitude of the inhibition has been 2B4 can be assessed from the sum of the products of theshown to be a function of the concentration of the b stock rate constants for reduction (phases 2–4) and the pro-5

solution from which the protein is added[28,34]. This was portions of P450 reduced in the respective phases[41]attributed to the fact that the b readily self-associates to (Table 3). The data conclusively show that the rate of5

form an octamer in concentrated solutions[28,46].Thus, it P450 reduction was decreased in the presence of either b5

was speculated that the higher order aggregate interfered or Mn b . The effect can be explained by a significant5

with the interaction of P450 reductase and P450 and thus decrease in the rate constant of all of the phases of P450impaired reduction of the latter[28]. Dilution of b stock reduction in addition to a reduction in the proportion of5

solutions (to allow for dissociation of octamers) before P450 reduced in the fastest phase (phase 2 in the table).adding b to the P450-mediated reactions resulted in both a This is slightly different from reports with the microsomal5

recovery in the rate of NADPH oxidation and stimulation system that showed b -related decreases in phase am-5

of reactions that were previously inhibited when b was plitude with minimal effects on the corresponding rate5

added from concentrated solutions[28,34]. In order for the constants[47]. However, these differences may be attribu-inhibitory effect to be eliminated, the protein had to be ted to those associated with the multi-lamellar membranediluted to 0.2mM. In the experiments shown inTable 2, system and the micellar reconstitution system. The abilitythe ‘‘concentrated’’ b and Mn b were added from stocks of 7EFC to stimulate the rate of P450 reduction also5 5

that were greater than 2mM. As a result, the observed seemed to be diminished in the presence of b and Mn b .5 5

reduction in the rate of NADPH oxidation could be The inhibitory effect of Mn b was greater than that of b5 5

attributed to the initial concentration of stock solutions as the net rate of reduction of P450 with Mn b was less5

used. than or equal to 60% of that with b , both with and without5

The reason for the decrease in the rate of NADPH substrate (7EFC). It cannot be determined if this resultsoxidation in the presence of Mn b is not clear. From our from a difference related to Mn b or if the system was5 5

data, the phenomenon could be explained on the basis of farther from equilibrium (the inhibitory effects of theincreased stabilization of the oxyferrous intermediate. This concentrated b solutions have been shown to mitigate5

would, in turn, explain the Mn b -mediated improvement after prolonged incubations with P450, reductase, and5

in reaction coupling. Alternatively, as proposed previously DLPC[33]) at the time of the assay relative to that with[28], it may be attributed to impairment of P450 reductase- b . The effects of concentrated solutions of b on P450-5 5

catalyzed reduction of the P450. mediated metabolism have been shown to diminish withDemonstration that the Mn b stabilizes the oxyferrous time as the mixture equilibrates[34]. Our results indicate5

J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275 271

T able 3aComparison of the effects of b and Mn b on the anaerobic reduction of P450 2B4 by reductase5 5

c dCondition Phase 1 Phase 2 Phase 3 Phase 4 ok * fraction (%)n n21 21 21 21k s k s k s k s1 2 3 4

b(%6S.D.) (%6S.D.) (%6S.D.)

Control 70 4.49 0.53 0.02 1.2 (100)(2062) (5166) (2967)

1Mn b 70 1.44 0.17 0.02 0.31 (26)5

(1263) (83617) (562)1b 70 2.82 0.37 0.03 0.50 (42)5

(1264) (3661) (5265)17EFC 70 5.88 1.45 0.02 3.16 (312)

(5666) (3063) (1467)17EFC1Mn b 70 2.63 0.50 0.02 0.53 (44)5

(1161) (4761) (43611)17EFC1b 70 3.14 0.35 0.031 1.34 (112)5

(3963) (33613) (28611)a The stopped flow spectrophotometry was performed as described in Materials and methods. Equimolar concentrations of P450 reductase, P450 2B4,

and b (or Mn b ) were reconstituted for the experiments as indicated. The control sample represents a sample without substrate that contained only5 5

reconstituted P450 and P450 reductase. The kinetic models used to fit the data resulted in root mean squared differences less than or equal to 0.0005.b The average of the percentage of the change in amplitude relative to the total change resulting from P450 reduction ((A /(A 1 A 1 A 1 A )3 100)n 2 3 4 5

for triplicate scans under each condition. The standard deviations (S.D.) of the amplitudes of triplicate kinetic scans for each of the conditions weredetermined from the variation that resulted when the rate constants for the replicate scans were fixed in the modeling program (described in Materialsandmethods).

c This value represents the summation of [k * (proportion reduced in phase 2)1k *(proportion reduced in phase 3)1k * (proportion reduced in phase2 3 4

4)] and has been used to quantitate the total rate of reduction of the P450 enzyme[41].d The percentage value is relative to the summation of the control sample.

that the lower rate of hydrogen peroxide formation in the oxidation. Increasing Mn b concentration from a 1:1 to a5

presence of Mn b (Table 2) may be attributed to a lower 2:1 ratio further decreased the rate of NADPH oxidation.5

rate of reduction although the data do not rule out the However, under these conditions, product formation waspossibility that the oxyferrous intermediate is also more also inhibited somewhat so that reaction coupling de-stable. creased from that observed with a 1:1 ratio. As seen in

Table 2,the rate of excess water formation appears to be3 .4. Effects of Mn b and b when the proteins are increased by the presence of Mn b as the rate increased5 5 5

added from diluted stock solutions with the concentration of Mn b5.

An aspect that is not explained by the previous experi-ment is the fact that addition of a ‘concentrated’ solution 4 . Discussionof Mn b inhibits the rates of NADPH oxidation and5

hydrogen peroxide production but has little effect on the 4 .1. Effect of Mn b on turn-over of aminopyrine by5

rate of product formation. It seems possible that if ‘dilute’ P450 2B4Mn b were added to the P450-catalyzed reactions,5

NADPH oxidation might not be inhibited and, in turn, As we showed previously, cytochrome b stimulated the5

product formation may be stimulated. The effect of ‘dilute’ P450 2B4-mediated metabolism of all of the substratesMn b on the metabolism of aminopyrine also is shown tested except benzphetamine[9]. In support of our hypoth-5

Table 2. esis that the b -mediated stimulation of aminopyrine5

When dilute Mn b was added, NADPH oxidation and metabolism was effected through a stimulatory conforma-5

reaction coupling were essentially similar to the control. tion change, b resulted in a dramatic reduction in theK5 d

The results when concentrated Mn b was added at a 1:1 of reductase and P450 2B4 binding in the presence of this5

ratio to P450 are similar to those inTable 2as NADPH substrate. In order to prove that this effect was responsibleoxidation was inhibited but reaction coupling was im- for the b -related stimulation and that it occurred through a5

proved. By comparing the coupling of reactions with Mn b -mediated change in P450 conformation, we used the5

b to those of the controls in the various experiments redox inactive Mn b to test whether a conformational5 5

(Table 2), it can be concluded that reaction coupling with mechanism could explain b -related stimulation of P450-5

Mn b is inversely related to the rate of NADPH oxidation. mediated aminopyrine metabolism. Mn b improved the5 5

Interestingly, product formation from aminopyrine was coupling for the metabolism of both 7EFC and amino-relatively unaffected by variations in the rate of NADPH pyrine by P450 2B4. Despite this effect on reaction

272 J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275

coupling, the Mn b did not stimulate product formation degree of reaction coupling will be referred to as ‘the5

with either substrate. coupling trend associated with Mn b ’.5

4 .2. Changes that affect reaction coupling in the 4 .3. A conformational equilibrium may regulatepresence of Mn b do not affect the rate of product progression through the P450 catalytic cycle5

formationWe propose that the P450 catalytic cycle is actually as

Our data may indicate that the Mn b results in follows: (1) the reductase reduces the ferric heme of the5

stabilization of the oxyferrous intermediate and thus P450 with one electron; (2) the resulting ferrous hemeimproved reaction coupling. More specifically, the inhibi- binds to molecular oxygen (this step has been shown totion of NADPH oxidation in the presence of b or Mn b occur rapidly[21]); (3) a change occurs in the P4505 5

could be attributed to less futile oxidation of NADPH conformation; (4) the second electron of the catalytic cycleduring the turn-over of the substrate or to a slower rate of is delivered to the oxyferrous intermediate; (5) heterolyticreductase-mediated first electron reduction of P450. Previ- or homolytic cleavage of the oxygen–oxygen bond occursous studies with purified P450 2B4 have shown that the with the formation of the putative perferryl intermediatehydrogen peroxide formed during metabolism by this [48]; (6) a second conformation change occurs; and (7)enzyme is almost entirely attributable to the dismutation of substrate is oxidized. Step 4 is generally thought to be thesuperoxide[28,45]. Stopped flow spectrophotometry was rate-limiting step of P450 2B4 catalysis[10]. However, inperformed to measure the rate of first electron reduction of our model, the putative conformation change (step 3)P450 (which is equivalent to the rate of formation of the actually may be the rate-limiting step. Our model requiresoxyferrous intermediate). Our findings show there is no the conformation of the P450 must change before steps 4evidence to support the possibility that the oxyferrous and 7 of the cycle can proceed. If an electron is deliveredintermediate of P450 is stabilized by Mn b . In fact, the before the P450 conformation changes, the P450 reaction5

decrease in the rate of reduction of P450 was more than becomes uncoupled forming superoxide and water, respec-sufficient to account for the decrease in hydrogen peroxide tively. We postulate that there is an equilibrium betweenformation. Furthermore, when the Mn b stock solution these catalytic conformations of P450. The relative propor-5

was diluted so that the rate of NADPH consumption was tion of each of the conformations is influenced both bynot inhibited, changes only were observed in the rate of substrates and the oxidative state of the heme iron (Fig. 1).hydrogen peroxide formation and not in the rate of Support for our model can be found in an elegant studyaminopyrine turn-over. If Mn b was stabilizing the examining freeze-trapped intermediates of P450cam using5

oxyferrous intermediate, hydrogen peroxide formation also monochromatic X-ray diffraction techniques[49]. Thisshould have been reduced when the Mn b was added from work showed that distinct protein conformers were associ-5

a dilute solution. Our findings show that reaction coupling ated with the different steps of the catalytic cycle. Inwas actually lower when the Mn b was diluted before addition, three catalytically significant conformations have5

adding to the reconstitution mixture. Thus, if our data been implicated in studies with P450 2B4[50] and theindicate increased stabilization of the oxyferrous inter- Asp251Asn mutant of P450cam[51]. Furthermore, in themediate, the effect is restricted to the aggregated and not study with the mutant of P450cam[51], the three con-the monomeric form of Mn b . Because this possibility formations were observable by Raman absorbance spec-5

seems unlikely, we feel an alternative explanation is troscopy only when reduced putidaredoxin was presentrequired. with the enzyme. These results could be interpreted to

If the delivery of the second electron of the catalyticcycle is the rate-limiting step of the reaction, the rate ofproduct formation is approximately equal to this step.

Because product formation was not affected by the con-centration of the Mn b stock solution, we can assume that5

the conditions that dramatically altered the rate of firstelectron transfer had no effect on the rate of secondelectron transfer. It seems independent processes regulateFig. 1. Schematic diagram representing an equilibrium between cata-these two steps. In the paragraphs below, we put forth thelytically significant conformations of P450 2B4. The arrows represent thehypothesis that the two steps may be related to two direction of change in the conformational equilibrium upon formation of

the different heme intermediates during the catalytic cycle. Conformationdifferent conformations of P450 and that the rate of the‘A’ represents a conformation stabilized by the ferric heme; conformationconformation change may be hindered when reduced (but‘B’ represents one that is stabilized by the oxyferrous heme; and

not oxidized) reductase is bound to the P450. For simplici- conformation ‘C’ represents one that is stabilized by the perferryl species.ty in the remainder of the discussion, the observed inverseThe rate of change between the conformations is also influenced by therelationship between the rate of NADPH oxidation and the substrate.

J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275 273

show that the reduced putidaredoxin was inhibiting the rate 4 .5. Model explaining the coupling trend associated withof change of the enzyme conformations. Mn b5

4 .4. Binding of reduced reductase inhibits P450 Using this model, we can now explain the couplingconformation changes trend associated with Mn b . The putative conformation5

change regulating the progression of the cycle before theIt is hypothesized that the aggregated Mn b inhibits the delivery of the second electron is inhibited as long as5

rate of electron delivery by the reductase but not the rate of reduced reductase and P450 are bound. Because the rate ofthe putative conformation change. As a novel perspective, electron transfer is inhibited by aggregated b , coupling is5

we propose that the rate of conformational equilibrium is improved because the oxyferrous intermediate does notinhibited when reduced (but not oxidized) reductase is wait as long to be reduced with the second electron (whenbound to the enzyme-substrate-b complex. Our model reductase and P450 disassociate). When dilute Mn b is5 5

distinguishes the effects of reduced and oxidized reductase used, the rate of electron transfer to the P450 is relativelybecause it has been shown previously that changes in P450 rapid. However, in this case, the oxyferrous intermediateconformation, which are induced by substrates and in- has a greater chance of degrading to release superoxidefluence the rate of reduction, occur very rapidly (during the before the requisite conformation change can occur.dead time of a stopped flow spectrophotometer) in thepresence of oxidized reductase[25]. Thus, the model

4 .6. Binding by b may inhibit the conformation change5requires the following sequence of events: (1) a substrate-required to turn over substrate

induced P450 conformation change when the P450 isbound to oxidized reductase during the dead time of a

Although the amount of excess water formed as a resultstopped flow spectrophotometer (this conformation change

of P450 catalysis cannot be determined definitively unlesswould correspond to a shift in the equilibrium towards that

the rate of oxygen consumption is measured, our datafavoring reduction of the ferric heme, and the magnitude of

suggest that the rate of this product may increase in thethe change in equilibrium would vary with different

presence of Mn b . Excess water results from reaction5substrates); (2) reduction of the P450 reductase while it isuncoupling at an alternative branch site of the catalytic

bound to the P450 (after reduction of the reductase, P450cycle [65]. The association of Mn b and increased excess5conformation changes would be inhibited); (3) reductionwater could be explained by a second catalytically signifi-

only of the P450 enzymes that were in the conformationcant conformation change (step 6 in our model). Thus, the

favorable to ferric heme reduction; (4) dissociation ofbinding of b may inhibit the second conformation change5partially reduced reductase and oxyferrous P450; and (5)necessary for the perferryl intermediate to oxidize sub-

conformation change and reduction of the remaining P450strate. If this is the case, it must be explained why many

enzymes. If the rate of dissociation of the reduced reduc-studies have shown that b reduces the excess water in5tase and P450 complex is different for the differentP450 reaction stoichiometry[28,66]. The idea of Perret

conformations of P450, the reduction of the remainingand Pompon[13] is invoked which proposes that oxidized

P450 would be multi-phasic. This possibility is tested inb can prevent excess water formation by binding to the5the following manuscript[67]. We believe the hypothesis isferric P450 and oxidizing the latter when it is reduced by

plausible because several studies have now providedP450 reductase. Mn b is redox-inactive, so it does not5compelling evidence that the conformation of reducedhave the capability to oxidize the reduced perferryl inter-

reductase is different from that of oxidized reductase[52].mediate.

It is likely that the oxidized reductase is most stable in aconformation in which the FAD and FMN domainsassociate[53]. This would allow reduction of the oxidizedprotein when bound to P450 by the hydrophobic inter- 5 . Conclusionaction site that controls the formation of the reductase–P450 complex [54–57]. For the reductase to transfer Our work provides a new perspective with respect toelectrons to the P450, the FAD-binding face of the FMN catalysis by P450 and the mechanism of action of cyto-domain must rotate on a hinge domain, away from the chrome b . As will be shown in the following manuscript5

FAD domain, and bind to a site on the proximal face of the [67], the model postulating that bound and reduced reduc-P450[58,59].Perhaps this occurs upon reductase reduction tase inhibits the putative conformation change(s) of P450and corresponds to the second putative electrostatic[60– 2B4 can be used to explain the multi-phasic reduction of62] (or hydrophilic [63,64]) site of interaction between P450[25]. We will test this possibility and providereductase and P450. Thus, we speculate that when the evidence that reduced reductase binds to P450 differentlyFMN domain of reductase is bound to the P450, the from oxidized reductase and that at least three conforma-changes in the catalytically significant conformations of tions are involved in the reduction of the enzyme by P450the P450 may be inhibited. reductase. Thus, the subsequent work provides support for

274 J.R. Reed, P.F. Hollenberg / Journal of Inorganic Biochemistry 97 (2003) 265–275

[26] L .D. Gruenke, J. Sun, T.M. Loehr, L. Waskell, Biochemistry 36the major constraints of our model involving the regulation(1997) 7114–7125.of P450 catalysis via a conformational equilibrium.

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