1

Cytochrome P450 Catalytic Mechanisms II P450 Cycle Stoichiometry and Decoupling: Overall cycle stoichiometries can be estimated from a consideration of the elementary reactions together with measurements of changes in substrates and products: 1. “Substrates” consumed are: NADPH, O2 and RH 2. “Products” formed during the catalytic cycle are (H2O, O2

-, ROH, NADP, H2O2) (note: only water cannot be measured). 3. Only “fully coupled turnover provides product ROH.

+3Fe NNS

Cys

OH H

Fe NNS

Cys

O

.++4 Fe NNS

Cys+3

RH

RH

+2Fe NNS

Cys

Fe NNS

Cys

OO

+2

RH

RH

+3Fe NNS

Cys

OO-

RH

ROH

H2O2

H2O

e

O2

Reducatse

Reductasealso B5 in microsomes?

e

2H +

2H +; 2e

2H +

H2O

RH

1 NADP + 1 H2O + 1 ROH1 NADPH + 1 O2 + 1 RH

1 NADPH + 1 O2 1 NADP + 1 H2O2

2 NADPH + 1 O2 2 NADP + 2 H2O

Stoichiometries for P450 cam (exlcudes superoxide)

O2-

-

C

D

2

We will look first at the cycle for CYP101 (P450 cam) with alternative substrates. CYP101 is a soluble P450 that uses an FAD/Putaredoxin electron donation system and NADH as the electron source. Note: the rate of superoxide formation in P450 cam is very slow so we will ignore it. ) 1. The CYP101 cycle is highly coupled with the “natural substrate” camphor. 2. Any branching to alternative products will reduce product formation rates from the optimal rate (totally coupled). How significant is this? Coupled and uncoupled turnover of substrates by P450 cam (wild type)

O O

5

6

Camphor Norcamphor3

Ethyl benzene Consumed Produced Substrate for P450cam

NADH Oxygen R-OH H2O2 H2O*

Camphor amounts

318 280 290 20 0

Norcamphor amounts

577 354 88 89 189

Ethylbenzene rates

52 46 5 40 6

Loida, P and Sligar, S. Biochemistry 32 11530 (1993) Atkins, W. and Sligar, S. Biochemistry 27 1610 (1988) *We have corrected for the first water molecule that is formed via the conventional monooxgenase pathway to produce compound I from the hydroperoxy species. The value for water formation reported reflects the reduction of compound I to water. Observations: 1. For the “natural substrate” camphor the cycle is almost fully coupled. Only a small amount of hydrogen peroxide is formed. Also there is no water beyond what is made during productive turnover. 2. For nor-camphor we see that the cycle goes to completion much less often (88/(88+89+189)= 24% coupled. The dominant pathway for norcamphor cycle is the reduction of compound I to water (52% of total cycles). 3. For ethylbenzene the dominant pathway is the formation of hydrogen peroxide (78%). The cycle only goes to completion 10% of the time! 4. Overall decoupling is observed and the contribution of the branching pathways is substrate dependent. Decoupling decreases enzyme efficiency and produces reactive oxygen species.

3

Systematic studies to study the linkage between substrate turnover, enzyme structure and products have been carried out: 1. Series of closely related substrates with well defined enzymes like CYP101 and their mutants in the hope that we can begin to understand how substrate structure influences decoupling. The effects of any particular mutation are unique to the enzyme-substrate fit and may diverge with different substrates… the multistep catalytic mechanism is complex, and there is still little information about what the parameters kcat and Km really represent in the case of this P450 and different mutations may affect different steps. Parikh, et al Biochem 38; 5283 (1999). 2. The ultimate test of these types of empirical studies is to develop predictive models of substrate oxidation rates. In order to understand and predict ROH formation rates we will need to understand the factors that promote or de-promote the oxidase pathways. Loida, P and Sligar, S. Biochemistry 32 11530 (1993) looked at the turnover of the “un-natural” substrate ethylbenzene by CYP101 wild type and mutants.

4

5

We will also have to understand how substrate structure affects (a) total turnover (b) decoupling (c) enzyme spin states and what substrate features promote/depromote oxygen insertion into substrates. Sibbesen, O, Zhang, Z. and Ortiz de Montellano. P. Arch. Bioch. Biophys. p 285 (1998)

SAR studies on the mammalian forms: This may not be easy as we are interested in rates, sites of oxidation and catalytic efficiency. Even when we have crystal structures of the mammalian forms. Effects on kcat and catalytic efficiency (substrate clearance) may not be meaninfully correlated

6

How will we eventually rationalize structure and function to predict affinities, products and rates?

The effects of any particular mutation are unique to the enzyme-substrate fit and may diverge with different substrates… the multistep catalytic mechanism is complex, and there is still little information

7

about what the parameters kcat and Km really represent in the case of this P450 and different mutations may affect different steps. Parikh, et al Biochem 38; 5283 (1999). Factors that appear to be important: 1. Ability of substrates to promote the high spin form (dehydration of the active site)? 2. Distance between the heme iron and site of oxidation on the substrate? 3. “Ease of oxidation” at the site of oxidation? 4. Ability of the substrates to inhibit decoupling reactions or stabilize productive cycle intermediates? 5. Ability of substrates to promote slow steps in the cycle (second electron transfer)? The role of cytochrome b5 in microsomal oxidation reactions represents a serious barrier to predictivity.

Stimulatory effects of cytochrome b5: See Pharmacol. Therap. 97 139-152 (2003) for a review Cytochrome B5 is a small heme containing protein that donates (ferrous B5) and accepts (ferric b5) a single electron. There is ample evidence that some P450 enzymes will form a high affinity complex with b5. Antibodies to B5 inhibit the oxidation of a number of substrates in intact microsomes. The implied stimulation of P450 activity by B5 has been demonstrated in reconstituted lipid vesicles of the enzymes as well as membranes prepared from standard expression systems. The effect of b5 is paradoxical in that it is: (1) substrate and enzyme dependent (2) not uniformly observed but inhibition is rarely observed (3) occasionally reported to be obligatory for some substrate enzyme pairs (4) stimulation of activity is also observed by with redox-silent apo-b5. Two major hypothesis (with many variations) have been forwarded to explain the B5 effect. 1. Electron transfer/enhanced coupling: Introduction of the second electron in the P450 cycle from b5 enhances the rate of second electron transfer observed with reductase alone. Supporting evidence includes reduction in superoxide formation in the presence of b5. decoupling to superoxide and/or enhancing the rate of second electron transfer.

D

C

-

O2-

RH

H2O

2H +

2H +; 2e

2H +

e

Reductasealso B5 in microsomes?

Reducatse

O2

e

H2O

H2O2

ROH

RH

-

Fe NNS

Cys

OO

+3

RH

RH

+2Fe NNS

Cys

OO

Fe NNS

Cys+2

RH

RH

+3Fe NNS

Cys

+4 .+Fe NNS

Cys

O

Fe NNS

Cys

OH H

+3

8

2. An allosteric effect where the structure of P450 is altered when complexed with b5. This is supported by the effect of apo-b5. A conformational change in the P450 could alter the substrate binding pocket of P450 in a manner that decreased uncoupling reactions thereby increasing the concentration of the active oxygen or by enhancing the rate of reaction of substrate with the active oxygen by decreasing the mean heme-substrate distance. Consider the rather confusing titles of two recent articles by Lucy Waskell and co-workers. Zhang H, Im SC, Waskell L. J Biol Chem. 2007 Oct 12;282(41):29766-76. Epub 2007 Aug 10. Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4. Zhang H, Hamdane D, Im SC, Waskell L. J Biol Chem. 2008 Feb 29;283(9):5217-25. Epub 2007 Dec 17. Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4. Quite recently studies of drug metabolism in mice where cytochrome b5 was conditionally knocked out were carried out by C. Roland Wolf and co-workers. Panel A shows the expression of b5 in various tissues in wild type and HBN (liver b5 knockout) animals. Panel C shows the amounts of various enzymes of interest in the livers of control and knockout animals. (JBC 283; 31385 (2008))

Table 1 shows the kinetics of oxidation of P450 substrate probes by microsomes from control and knock-out animals.

9

Table 2 shows the effects of b5 knockout on oral and iv pharmacokinetics of test drugs in vivo in mice. In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.

10

CYP2B4 – CPR CYP2B4 – b5

the reaction are followed spectrophotometrically by observing thespectral changes that occur as a result of changes in the redox stateof the interacting proteins. Within the dead time of the instrument,oxygen rapidly binds to the ferrous cytochrome P450. The oxyfer-rous cytochrome P450 immediately accepts an electron (the sec-ond electron required for catalysis; recall the first electron wasprovided by dithionite) from the redox partner and undergoescatalysis resulting in product formation and regeneration of theferric protein (Fig. 1). Alternatively, uncoupling of the reaction willoccur with hydrogen peroxide and ferric protein formation. Themore rapid formation of the ferric cytochrome P450 in the pres-ence of a competent redox partner was considered to representthe rate of product formation. Control studies determined the rateof autoxidation of each protein in the absence of a redox partner.Meanwhile the redox partner has undergone oxidation after donat-ing the electron. Cytochrome b5 reverts to the ferric protein and thereductase becomes the semiquinone form, neither of which can re-duce the newly formed ferric cytochrome P450. Hence, the cyto-chrome P450 turns over only once. A similar protocol was alsoemployed in rapid chemical quench studies and freeze quenchEPR studies to be described later in this article [28,29].

Fig. 7 compares the kinetics of formation of the ferric cyto-chrome P450 (followed at 438 nm) from the oxyferrous species inthe presence of cytochrome b5 (k = 11 s!1) and cytochrome P450reductase (k = 0.09 s!1). Remarkably, oxyferrous cytochrome P450decayed to the ferric enzyme approximately 100-fold faster in thepresence of cytochrome b5. Fig. 7 and Table 2 demonstrate thatcytochrome b5 (k = 9.3 s!1) and cytochrome P450 (k = 10.5 s!1) oxi-dize simultaneously to the ferric proteins. In contrast, Fig. 7 and Ta-ble 2 demonstrate that reductase oxidizes with a rate constant of8.4 s!1 while cytochrome P450 forms ferric cytochrome P450approximately 100-fold more slowly, with a rate constant of0.09 s!1 which is the rate of catalysis under steady-state conditions(kcat = 0.08 s!1) at 15 !C, the experimental temperature. Note thatthe reductase and cytochrome b5 undergo spectral changes whichreflect donation of an electron to cytochrome P450 at essentiallythe same rate. Surprisingly, cytochrome P450 behaves differentlyin the presence of its two redox partners. These data were inter-preted to indicate that in the presence of cytochrome P450 reduc-tase catalysis by cytochrome P450 occurs via a long-livedintermediate. Global analysis of the spectral data obtained withthe photodiode array detector was unable to detect a new spectralintermediate. Quantitative analysis of the reaction mixture by LC–

MS/MS revealed that the product, norbenzphetamine, was formedwith a coupling efficiency of 52% with cytochrome b5 versus 32%with the reductase. Considered as a whole, the results indicate thatin the presence of reductase, a relatively stable, reduced oxyferrouscytochrome P450 intermediate is formed and that the rate-limitingstep in catalysis is not introduction of the second electron butrather a later step in the catalytic cycle. It is hypothesized that cyto-chrome b5 and reductase are effectors of cytochrome P450 that in-duce different conformational changes in the active site uponbinding. In view of the instability of the known reduced oxyferrousspecies (peroxo and hydroperoxo in Fig. 1), it is remarkable that oneof them is stabilized under ambient conditions. It is also possiblethat proton delivery is slower because of suboptimal organizationand function of the essential proton delivery machinery.

Catalysis is faster in the presence of cytochrome b5 under singleturnover conditions

Because the spectrally measured rate of decay of oxyferrouscytochrome P450 to the ferric protein in the presence of its redoxpartners was multiphasic, it was not possible to unambiguouslydetermine when product was formed. For this reason the rate ofproduct formation was measured directly using a rapid chemicalquench technique [28]. The reaction was conducted as describedfor the spectrophotometric studies except that the reaction wasquenched at designated times and product formation measuredby a sensitive LC–MS/MS assay capable of detecting low picomolequantities of the product, norbenzphetamine. A prereduced com-plex of equal amounts of cytochrome P450 with either cytochromeb5 or reductase was mixed with an oxygen-containing buffer. Thereaction was allowed to proceed and samples were collected as afunction of time. Fig. 7 demonstrates that the rate constant for for-mation of norbenzphetamine (product of benzphetamine metabo-lism) was within experimental error identical to the rate constantof the formation of the ferric cytochrome P450 in spectrophoto-metric experiments. The results also establish that benzphetamineis metabolized approximately 100-fold more quickly in the pres-ence of cytochrome b5 than in the presence of reductase. Con-versely, metabolism is 100-fold slower in the presence ofreductase than cytochrome b5. Metabolism of a second substrate,cyclohexane, is also slower and less efficient in the presence ofreductase, demonstrating the different effects of the redox partnerson cytochrome P450 2B4 activity is not dependent on the sub-strate. In aggregate, our experiments under single turnover condi-tions with preformed reduced complexes of cytochrome P450 2B4showed that cytochrome b5 and reductase catalyze product forma-tion monophasically, with easily distinguishable rate constants fortwo substrates, benzphetamine and cyclohexane.

Why catalysis is slower when the second electron is providedby cytochrome P450 reductase is not understood at the presenttime. Understanding the biochemical mechanism by which thedrug metabolizing cytochromes P450 catalytic rate and efficiencyis modulated by its redox partners should provide insights whichwill assist in harnessing its strong oxidative power for a numberof practical applications.

The observation that the two redox partners support catalysis atsuch markedly different rates raises an intriguing question: what isthe basis of this distinctive behavior of oxyferrous cytochromeP450 in the presence of its two electron donors? Examination ofthe cytochrome P450 catalytic cycle (Fig. 1) reveals that, in theory,either the peroxo species (Fe3+OO)2!, its protonated form, thehydroperoxo intermediate (Fe3+OOH)!, an oxidized substrate–heme complex or a previously unidentified ephemeral compoundcould be the reduced oxyferrous transient species. A hydroperoxospecies would be more stable than the very nucleophilic peroxo

Fig. 7. Comparison of the kinetics of the decay of oxyferrous cytochrome P450 tothe ferric species and of norbenzphetamine formation in the presence of substrate,benzphetamine. The DA438nm represents the decay of the oxyferrous P450 2B4 tothe ferric protein [22]. The data for product formation are from [28]. ( ), productformation by P450–b5; ( ), product formation by P450–CPR; ( ), DA438nm forP450–b5; ( ), DA438nm for P450–CPR.

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species which is protonated at 70 K but not 4 K in cytochromeP450cam [49]. In fact, preliminary freeze quench EPR data has beenobtained indicating that a hydroperoxo intermediate accumulatesunder single turnover conditions in the presence of reductase. Ifthis is true, it follows that delivery of the second proton to thehydroperoxo species is delayed, presumably by a conformationalchange induced in the active site by reductase binding on the prox-imal surface of cytochrome P450 (Fig. 5). In some manner, the pro-ton delivery machinery has been temporarily disrupted.

Under single and multiple-turnover conditions, cytochrome b5and reductase compete to provide the second electron requiredfor cytochrome P450 catalysis

Establishing distinctive rates at which cytochrome b5 andreductase support catalysis under single turnover conditions hasenabled us to determine whether cytochrome b5 or reductasemediates catalysis when both redox partners are present and togain a better understanding of how cytochrome b5 influences catal-ysis under steady-state, multiple-turnover conditions [28]. Byvarying the ratio of cytochrome b5 to reductase present in the reac-tion mixture, it has been possible to demonstrate that these tworedox partners compete for the binding site on the proximalsurface of cytochrome P450 2B4. In the presence of equimolarconcentrations of cytochrome P450, cytochrome b5, and reductase,the formation of product was biphasic and occurred with fast andslow rate constants diagnostic of catalysis by cytochrome b5 andreductase respectively. At the 1:1:1 M ratio, 32% of the productcan be attributed to cytochrome b5 whereas 68% representsproduct formation by reductase. Because of a lengthy overnightpreincubation of the three proteins prior to the experiment, itwas concluded that under our experimental conditions reductasehad a higher affinity for cytochrome P450 than cytochrome b5. Ahigher molar ratio of cytochrome b5 enhanced the amount of prod-uct generated by cytochrome b5 and decreased the quantity ofproduct formed by reductase. At a 3-fold molar excess, cytochromeb5 generated 74% of the product, while the remainder was contrib-uted by the reductase.

Recall that under single turnover conditions the first electron isprovided by dithionite, not reductase. Under steady-state condi-tions the situation is more complicated, due to the fact that onlyreductase possesses a redox potential capable of delivering an elec-tron to the ferric cytochrome P450. Cytochrome b5 cannot providethe first electron, only the second. Using published data from stea-dy-state experiments performed in our laboratory, Table 3 demon-

strates the effect of increasing the molar ratio of cytochrome b5 onNADPH consumption and formaldehyde formation from the N-demethylation of the substrate, benzphetamine [28]. At molar ra-tios equal to or greater than 1, cytochrome b5 inhibits NADPH con-sumption and product formation. However, it stimulates productformation and enhances reaction efficiency at molar ratios lessthan 1. Thus at molar ratios 61, cytochrome b5 enhances substratemetabolism but progressively inhibits NADPH consumption andmetabolism at higher cytochrome b5:reductase molar ratios. Thesystematic variation of the molar ratio of cytochrome b5 on theactivity of the purified reconstituted cytochrome P450 2B4 systemleads to the proposal that the stimulatory effect of cytochrome b5on catalysis is due to its ability to increase the catalytic rate andefficiency of NADPH coupling to product formation. The inhibitoryproperties of cytochrome b5 are a consequence of its ability tooccupy the reductase binding site on the proximal surface of cyto-chrome P450, thereby preventing the reductase from binding andproviding the first electron to ferric cytochrome P450. Formationof ferrous cytochrome P450 is an early critical step in the reactioncycle.

Cytochrome b5 inhibits reduction of ferric cytochrome P450 2B4

To examine our hypothesis that, under steady-state conditions,cytochrome b5 attenuated NADPH utilization and the activity ofcytochrome P450 2B4 by competing with reductase for its cyto-chrome P450 binding site and preventing reduction of ferric cyto-chrome P450, the rate of electron transfer was directly measured inthe presence of varying concentrations of cytochrome b5 and Mncytochrome b5. The reduction of ferric cytochrome P450 2B4 wasmeasured by determining the rate of formation of the ferrous

Table 2Summary of rate constants and amplitudes for autoxidation and redox reactions of cyt P450, cyt b5, and 5-deazaFAD reductase in the absence and presence of their redox partners.(Reprinted with permission of Am. Chem. Soc. Biochem. 42 (40) (2003).)

Syringe 1 Syringe 2 k (nm) obsa Species obsa Phase 1 Phase 2 Phase 3

A (%) k1 (s!1) A (%) k2 (s!1) A (%) k3 (s!1)

2E-reduced 5-deazaFAD reductasec O2 450585

ReductaseReductase

100 ± 11100 ± 8

0.007 ± 0.0010.007 ± 0.001

Cyt b2"5 O2 422 Cyt b5 97 ± 5 0.005 ± 0.0003

P4502+ O2 438 P450 25 ± 3 0.96 ± 0.2 34 ± 6 0.13 ± 0.04 41 ± 7 0.016 ± 0.005P4502+ + BPb O2 + BP 438 P450 40 ± 4 0.13 ± 0.05 60 ± 7 0.0480 ± 00.0042E-reduced 5-deazaFAD reductase cyt b2"5 422

567Cyt b5reductase

98 ± 10,100 ± 12

0.002 ± 0.00020.002 ± 0.0004

P4502+-cyt

b2"5 + BP

O2 + BP 422438

Cyt b5P450

50 ± 662 ± 7

9.3 ± 0.710.5 ± 1.5

4 ± 0.118 ± 1.1

0.43 ± 0.210.83 ± 0.18

46 ± 720 ± 3

0.005 ± 0.00030.005 ± 0.001

P4502+ – 2e-reduced 5-deazaFAD reductase + BP O2 + BP 598438

ReductaseP450

31 ± 6 8.4 ± 1.5 52 ± 686 ± 10

0.37 ± 0.060.090 ± 0.01

17 ± 0.714 ± 0. 5

0.041 ± 0.0050.0012 ± 0.002

Reprinted with permission of American Chemical Society, Biochemistry 42(40), 2003.a Observed.b 1 mM benzphetamine.c These data are from [22,48].

Table 3Effect of cyt b5 on the rate of NADPH consumption and benzphetamine metabolism bycyt P450 2B4 under steady-state conditions at 30 !C.

Molar ratio P450:CPR:b5 NADPH Formaldehydenmol/min/nmolof cyt P450

nmol/min/molof cyt P450

1:1:0 83 ± 5.5 47 ± 3.31:1:0.5 81 ± 2.1 56 ± 0.81:1:1 66 ± 3.2 48 ± 1.01:1:3 36 ± 6.3 30 ± 3.81:1:5 25 ± 1.3 21 ± 1.8

Data from: [28].

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FMN domains would be dramatically slower (410-fold) comparedto the wild-type enzyme.

Comparison of the kinetics of the reduction of oxyferrouscytochrome P450 and its decay to the ferric protein in thepresence of cytochrome b5 and cytochrome P450 reductase

There were two reasons we were prompted to measure the rateof delivery of the second electron to cytochrome P450. The firstwas the observation that cytochrome b5 increased the efficiency ofcatalysis in a reconstituted system under steady-state conditionsby decreasing superoxide formation [8,11,47]. The second was thesuggestion that cytochrome b5 mediated its effect by reducing oxy-ferrous cytochrome P450 faster than cytochrome P450 reductase,

allowing less time for autoxidation and superoxide formation.Examination of the cytochrome P450 reaction cycle (Fig. 1) demon-strates that oxidation of substrate requires two electrons and twoprotons. The first electron, which reduces the ferric protein, is deliv-ered by cytochrome P450 reductase. Cytochrome b5 cannot deliverthe first electron because of its high potential (+25 mV versusapproximately !245 mV for benzphetamine bound enzyme) [22].The second electron, which reduces oxyferrous cytochrome P450,canbedeliveredby either cytochromeb5 or cytochromeP450 reduc-tase. Cytochrome b5 is able to donate the second electron becausethe potential of the oxyferrous enzyme has increased to about+20 mV [39]. It was possible to directly measure both the rate ofreduction of oxyferrous cytochrome P450 2B4 and its rate of oxida-tion of reductase by utilizing the 5-deaza FAD T 491 V cytochromeP450 reductase instead of the wild-type protein. The advantage ofemploying the5-deazaFAD reductasewas that it underwent a singleredox reaction, oxidation of the FMN hydroquinone to a semiqui-none, whose spectral changes were readily interpretable [22,48].Because the T 491 V reductase mutant bound FAD less tightly thanthe wild-type protein, it was possible to exchange FAD for 5-deazaFAD, which does not undergo a change in redox state under ourexperimental conditions. The redox active critical N5 of FAD is re-placed by a carbon atomwhich renders the 5-deaza FAD essentiallyredox inactive but still able to bind to and maintain the structuralintegrity of the reductase. It was necessary to replace the FAD witha redox silent analogue in order to be able to unambiguously inter-pret the spectral changes that occur when the two electron reducedreductase transferred an electron to oxyferrous cytochrome P450.Since the distributionof electrons in this diflavinprotein is governedsolely by the reduction potentials of its cofactors, there are ninedifferentways electrons canbe distributed.Hence, nine possible un-ique forms of the protein exist. At any given level of oxidation, otherthan complete oxidation or total reduction,more than one species ofreductasewill exist. Stoichiometric addition of two electron equiva-lents to the 5-deaza FAD reductase by dithionite produced a twoelectron reduced reductase capableof transferringonlya single elec-tron from its FMN domain to its redox partners. The FMN semiqui-none is stable and does not donate an electron to acceptor proteins.

A brief description of the experimental protocol employed tomeasure the reduction of cytochrome P450 2B4 by 5-deaza FADT 491 V reductase under single turnover conditions follows. A sim-ilar protocol was also employed in subsequent experiments wherethe wild-type reductase was utilized. Single turnover conditionsrefer to the fact that the experiments were conducted under condi-tions in which a molecule of cytochrome P450 can generate, atmost, a single molecule of product. The strategy of this experimentis to bypass the first electron transfer step by reducing the proteinswith stoichiometric amounts of dithionite so that the reaction canbe initiated by mixing an oxygen-containing solution with asolution of the prereduced 1:1 cytochrome P450–redox partnercomplex. Cytochrome P450, its redox partner – either cytochromeb5 or reductase – along with substrate, phospholipid and buffer aremixed together and incubated in a glovebox to perform the desiredcomplex and remove oxygen. The preformed complex, 15–30 lM,consisting of equal amounts of cytochrome P450 and its redoxpartner is stoichiometrically reduced with dithionite. In the caseof the cytochrome b5 – cytochrome P450 complex, both proteinsare reduced to the ferrous state. When reductase is the redoxpartner, it is reduced to the 2-electron state, which can transferonly a single electron to cytochrome P450 since the one electronreduced FMN semiquinone is stable and not capable of donatingan electron.

The prereduced complex is placed in one syringe of the UV–visible stopped-flow spectrophotometer, which is housed in ananaerobic glove box, and subsequently rapidly mixed with theoxygen-containing buffer from a second syringe. The kinetics of

Fig. 5. Cytochrome P450–reductase model complex bound to a membrane. Thereductase is molecule A (PDB code 3ES9) of the open conformation of reductase.

Fig. 6. Cytochrome P450–reductase model complex. The complex was generatedwith pymol and accounts for available mutagenesis data. PDB code of P450 2B41SUO and molecule A PDB code 3ES9.

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yielded a cytosolic protein that did not bind to the endoplasmicreticulum, whereas lengthening the membrane anchor by 5 aminoacids caused the protein to be transported to the plasma mem-brane of COS cells [40,41]. In vitro the a-helical membrane span-ning domain has been demonstrated to interact with cytochromeP450 2B4 via non-specific interactions, not via a specific stereo-chemical knobs in socket fit [42].

Alanine was inserted by site-directed mutagenesis in the trans-membrane domain at six positions to ‘‘frame shift’’ the amino acidscarboxy terminus to it. Insertion of a residue to an a-helix causesall downstream residues to be rotated by !100!. All mutants wereas active as the wild-type protein. Whether the membrane anchorsof the two heme proteins interact in a membrane bilayer is notknown. It would depend on the relative affinity of the membraneanchors for one another compared to their affinity for the hydro-phobic lipids of the bilayer.

A model of the cytochrome P450 2B4–cytochrome b5 complex ispresented in Figs. 3 and 4, which accounts for the mutagenesis dataon both proteins. The cytochrome P450 2B4 binding site for cyto-chrome b5 has been identified on the proximal surface and de-scribed in ‘‘Cytochrome b5 and cytochrome P450 reductase bindto the basic, positively-charged, proximal surface of cytochromeP450 2B4 on unique but overlapping sites’’. It was expected thatacidic residues surrounding the heme of cytochrome b5 would beinvolved in the interaction with cytochrome P450. Amino acidssurrounding the heme have been mutated and tested for their abil-ity to bind and support the activity of cytochrome P450 2B4 (man-uscript in preparation). In order to determine which residues fromthe two proteins were in contact, a double mutant cycle experi-ment was conducted with all possible combinations of wild-typeand mutant proteins. In such cycles, the sum of free energy lossby the two single mutations is compared to the free energy lossby the double mutation. When the loss by the complex with thetwo mutant proteins is greater, the residues are considered to bein contact in the complex [43]. This experiment revealed thatAsp64 and Val65 of cytochrome b5 are in contact with Arg 122,R126 and Lys 433 of cytochrome P450 2B4 (Figs. 3 and 4).

Interaction of cytochrome P450 2B4 with cytochrome P450 reductase

Cytochrome P450 reductase is a membrane-bound diflavin pro-tein, which transfers two electrons sequentially from NADPHthrough FAD to the FMN cofactor, which is the ultimate donor of

electrons to cytochrome P450 and other acceptor proteins [4].The reductase is bound to the membrane by an amino terminala-helix. A linker of approximately 15 amino acids connects themembrane-bound domain to the soluble flavin containing domain.A crystal structure of the reductase has been determined in a con-formation appropriate for interflavin electron transfer, in whichthe two flavin rings are in contact [44]. However, cytochromeP450 cannot be docked to this conformation of the reductase in amanner that would allow physiologic electron transfer betweenthe proteins. Therefore, it has been proposed that the two flavindomains would separate in order to allow the FMN domain to dockwith cytochrome P450. The structure of a mutant with a 4-amino-acid deletion in a hinge connecting the FMN domain to the rest ofthe protein has been determined in three markedly extended con-formations in which the flavins are separated by 30–60 Å [45].These three very different conformations demonstrate that the en-zyme is able to undergo a structural rearrangement that separatesthe FAD and FMN domains, which allows the FMN to dock with itsredox partners. It is proposed that the wild-type enzyme under-goes an analogous conformational change in the course of shuttlingelectrons from FAD to acceptor proteins. To visualize a plausiblemovement, the FMN domain would undergo to transfer electronsfrom FAD to cytochrome P450 2B4, see http://www.molmovdb.org/cgi-bin/morph.cgi?ID=234,385-8941. The link shows a movieof energy minimized conformational changes the FMN domainwould experience in translating from its position in the wild-typeprotein to its position in molecule A, the least open conformationof the three conformations [46]. Figs. 5 and 6 show a model ofthe complex.

When provided with sufficient electrons, the mutant proteincan reduce both the ferric and oxyferrous proteins at the same rateas the wild-type protein. Reduction of the oxyferrous protein re-sults in product formation demonstrating that the open conforma-tion of reductase is functional. In view of the great distanceseparating the FMN and FAD domains in the open conformation,it is not unexpected that electron transfer between the FAD and

Fig. 3. Cytochrome P450 2B4–cytochrome b5 model complex bound to a mem-brane. Residues V66 and D65 of cytochrome b5 are in contact with R126, K433, R122of cytochrome P450 2B4. (PDB code of 1SUO).

Fig. 4. Cytochrome P450–cytochrome b5 model complex. The complex wasgenerated with pymol and accounts for the available mutagenesis data. PDB codeof P450 2B4 1SUO.

S.-C. Im, L. Waskell / Archives of Biochemistry and Biophysics xxx (2010) xxx–xxx 5

Please cite this article in press as: S.-C. Im, L. Waskell, Arch. Biochem. Biophys. (2010), doi:10.1016/j.abb.2010.10.023

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What is the normal ratio of CPR/b5P450 in HLM? CYP Content (pmol/mg) % Total CYP CPR/b5/CYP Ratio Total 300 1:3:3 3A 96 32 1:3:1 2E1 22 7.3 5:14:1 2D6 5 1.7 20:60:1 2A6 14 4.7 7:21:1 1A2 42 14 2:7:1 2C9 49 16 2:6:1 2C other 11 3.7 9:27:1 Things to consider: When it comes to b5 effects and prediction of rates and routes in vivo Roger MacNammee was right on. “what we know isn’t worth much and what we don’t know is really important”. 1. What is the optimal ratio to use in reconsititued and heterologously expressed P450 enzyme systems which seek to mimic the microsomal and in vivo situation? 2. Cytochrome b5 is normally reduced by a different enzyme, b5-reductase which uses NADH rather than NADPH as the source of electrons:

2 ferric b5 + NADH 2 ferrous b5 + NAD. Thus b5 supported activity in vivo and in hepatocytes will be different unless the full system and NADH are present. 3. The affinity of b5 for P450 enzymes is lower than reductase. 4. b5 appears to stimulate the metabolism of “poor substrates” more than “good substrates”.