Protonation of the Hydroperoxo Intermediate of Cytochrome P450 2B4

Oct 31, 2016 - *Department of Anesthesiology, University of Michigan, Mail Stop 151, 2215 Fuller Rd., Ann Arbor, MI 48109-0112. E-mail: waskell@umich...
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Protonation of the hydroperoxo intermediate of cytochrome P450 2B4 is slower in the presence of cytochrome P450 reductase than in the presence of cytochrome b5 Naw May Pearl, Jarett Wilcoxen, Sangchoul Im, Ryan C Kunz, Joseph E. Darty, R. David Britt, Stephen W. Ragsdale, and Lucy Ann Waskell Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00996 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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Protonation of the hydroperoxo intermediate of cytochrome P450 2B4 is slower in the presence of cytochrome P450 reductase than in the presence of cytochrome b5 Naw May Pearl1, Jarett Wilcoxen2, Sangchoul Im1, Ryan Kunz3, Joseph Darty4, R. David Britt2, Stephen W. Ragsdale5, Lucy Waskell1

1

Department of Anesthesiology, University of Michigan Medical School, Ann Arbor, MI, United

States 2

Department of Chemistry, University of California, Davis, Davis, CA, United States

3

Work done in Department of Biological Chemistry, University of Michigan; currently at

Department of Cell Biology, Harvard Medical School, Boston, MA, United States 4

Work done in Department of Biological Chemistry, University of Michigan; currently at

Siemens, A.G., Newark, DE, United States 5

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI,

United States 1|Page ACS Paragon Plus Environment

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ABSTRACT. Microsomal cytochromes P450 (P450) require two electrons and two protons for the oxidation of substrates. Although the two electrons can be provided by cytochrome P450 reductase, the second electron can also be donated by cytochrome b5 (b5). The steady-state activity of P450 2B4 is increased up to 10-fold by b5. To gain a greater understanding of the molecular basis of the stimulatory effect of b5 and to test the hypothesis that b5 stimulates catalysis by more rapid protonation of the anionic ferric hydroperoxo heme intermediate of P450 (Fe+3OOH)¯ and subsequent formation of the active oxidizing species (Fe+4OH+•), we have freeze quenched the reaction mixture during a single turnover following reduction of oxyferrous P450 2B4 by each of its redox partners, b5 and P450 reductase. The EPR spectra of the freezequenched reaction mixtures lacked evidence of a hydroperoxo intermediate when b5 was the reductant presumably because hydroperoxo protonation and catalysis occurred within the dead time of the instrument. However, when P450 reductase was the reductant, a hydroperoxo P450 intermediate was observed. The effect of b5 on the enzymatic efficiency in D2O and kinetic solvent isotope effect under steady-state conditions are both consistent with the ability of b5 to promote rapid protonation of the hydroperoxo species and more efficient catalysis. In summary, by binding to the proximal surface of P450, b5 stimulates the activity of P450 2B4 by enhancing the rate of protonation of the hydroperoxo intermediate and formation of Compound I, the active oxidizing species, which allows less time for side product formation.

ABBREVIATIONS: cytochrome P450 (cyt P450 or P450), cytochrome b5 (cyt b5 or b5), Dilauroylphosphatidylcholine (DLPC), cytochrome P450 reductase (cyt P450 reductase or P450 reductase or reductase or CPR), Compound I (oxyferryl cytochrome P450, Cpd I).

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Introduction The cytochromes P450 (cyts P450, P450) are a superfamily of heme-containing mixed-function oxygenases that occur in all kingdoms of life. They are responsible for the oxygenation of a staggering array of substrates. In humans, the 50 microsomal cyts P450 catalyze the oxidation of a variety of essential endogenous compounds, a majority of the exogenous compounds in our diet, and a large percentage of the drugs in use today.1,2,3 Rabbit cytochrome P450 2B4, the enzyme we are studying, is a microsomal protein that is ~ 78 % identical to the human microsomal, drug-metabolizing cyt P450 2B6. Cytochrome P450 catalyzes the reaction: SH + O2 + 2 electrons + 2H+ → SOH + H2O where S is the substrate. The two electrons are indirectly provided by NADPH via the diflavin (FAD and FMN) cytochrome P450 reductase. Because of its high redox potential (~ -25 mV), cytochrome b5 can only donate an electron to oxyferrous cyt P450, not the ferric protein.4,5,6,7,8 The catalytic cycle of cyt P450 is shown in Figure 1.

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Binding of substrate to cyt P450 increases the potential of cyt P450, which triggers reduction of the ferric protein (first electron transfer) by cyt P450 reductase to the ferrous protein. Oxygen readily binds to the ferrous heme, forming a moderately stable oxyferrous heme that is considered to be a ferric-superoxide species. It is the last step in the catalytic cycle that is readily accessible to study by spectroscopic techniques. A second electron is transferred to the oxyferrous heme from either cytochrome b5 or cytochrome P450 reductase, forming a transient +3

dianionic peroxo species (Fe OO)¯ which is protonated at 77 K forming the equivalent of a complex of the ferric iron with a hydroperoxide (Fe+3OOH)¯. The hydroperoxo species is also unstable in cyts P450. In cyt P450 2B4 the side chain of the conserved active site Thr 302 accepts a hydrogen bond from the transient hydroperoxo intermediate, which receives a second proton from the active site proton delivery network and rapidly undergoes spontaneous heterolytic oxygen bond cleavage, forming water and an oxyferryl (Fe+4 = O) π cation radical, Compound I.9 Compound I is the active oxidizing species. Investigators have been intrigued for many years by the observation from numerous laboratories that cytochrome b5 can stimulate, inhibit or have no effect on the catalytic activity of the membrane-bound, microsomal cytochromes P450. It was also puzzling that the effect of cyt b5 was dependent on the cytochrome P450 isozyme and substrate.10,11,12,13,14 We became interested in the problem because of the observation that cytochrome b5 markedly increased the metabolism of the poor substrate, the volatile anesthetic methoxyflurane (a methyl ethyl ether) by the membrane-bound, microsomal cyt P450 2B4, whereas under the same conditions, cytochrome b5 apparently did not have an effect on the oxidation of the extensively metabolized substrate, benzphetamine.5 Previous studies have demonstrated that, in the presence of cytochrome b5, more reducing equivalents from NADPH were utilized to generate metabolites 4|Page ACS Paragon Plus Environment

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of both substrates rather than the side products superoxide and hydroperoxide. In other words, cytochrome b5 increases the “coupling” of NADPH consumption to product formation by cytochrome P450 2B4.15 Presteady-state kinetic studies of the reduction of oxyferrous cytochrome P450 2B4 under single turnover conditions in a stopped-flow spectrophotometer demonstrated that, although cytochrome b5 and the reductase reduced oxyferrous cytochrome P450 2B4 at the same rate, the oxyferrous heme underwent a single catalytic cycle, considerably slower, in the presence of the reductase.5,16,17 These results indicated that reduction of P450 2B4 was not rate limiting but that a reaction following reduction of oxy ferrous cyt P450 2B4 was rate limiting and that catalysis in the presence of P450 reductase occurred via a semi-stable intermediate. In contrast, cyt b5 and the oxyferrous cyt P450 simultaneously converted to the ferric proteins without evidence of a detectable cyt P450 intermediate. To confirm that the spectrally measured rapid rate of decay of the oxyferrous protein in the presence of its redox partners represented catalysis, the rate of product formation was also measured under single turnover conditions following chemical quenching of the reaction at designated times. The timedependent formation of product was determined by a sensitive LC-MS/MS assay. Product was formed ~ 100 times faster in the presence of cyt b5 than in the presence of reductase at a rate similar to that predicted by the spectrophotometric studies. The coupling efficiency was 52 % with cyt b5 and 32 % with P450 reductase virtually identical to the results under steady-state conditions.17 Examination of the cyt P450 catalytic reaction cycle revealed that either the peroxo species (Fe+3OO)¯ or its protonated form, the hydroperoxo intermediate (Fe+3OOH)¯ could be the reduced oxyferrous species that transiently exists in the presence of the reductase (Figure 1). Cryoreduction EPR experiments at 77 K with cyt P450 2B4 and cyt P450 camphor have 5|Page ACS Paragon Plus Environment

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established that the dianionic peroxo compound is protonated at 77 K by an active site proton delivery network while the monoanionic hydroperoxo compound is stable under those conditions.18,19,20 These data prompted the hypothesis that cyt P450 reductase slows cyt P450 2B4 catalysis by delaying proton delivery to the hydroperoxo intermediate, which should, therefore, accumulate when the reductase but not cyt b5 reduces oxyferrous cyt P450 2B4. In this manuscript we report that using freeze-quench EPR studies, it has been possible to demonstrate that under ambient conditions a ferric hydroperoxo intermediate can be trapped during a single turnover in the presence of P450 reductase but not cyt b5. These results demonstrate that binding of b5 and P450 reductase on the proximal surface of P450 2B4 not only results in electron transfer but that each redox partner transmits unique structural information to the active site proton delivery network. Materials and Methods All chemicals used are ACS grade. Benzphetamine, sodium dithionite, methyl viologen, IPTG, and carbenicillin were purchased from Sigma Aldrich. Glycerol (for protein purification) was purchased from Fisher Scientific. Dilauroylphosphatidylcholine (DLPC) was purchased from Doosan Serdary Research Laboratory (Toronto, Canada). Redistilled glycerol (Molecular biology-grade) and protease inhibitor cocktails were purchased from Roche. ∆5-aminolevulinic acid was purchased from Research Products International Corp. C41 cells were purchased from Lucigen Middleton, WI. 30,000 MWCO centrifugal tubes were purchased from Millipore. Protein Expression and Purification Full length wild type rabbit cytochrome P450 2B4 (cyt P450 or P450), full length rat wild type NADPH cytochrome P450 oxidoreductase (CPR) and full length rabbit cyt b5 were expressed in

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Escherichia coli C41 (DE3) and purified as previously described.21,22,23 Each purified protein occurred as a single band on an overloaded SDS polyacrylamide gel. Cyt P450 2B4 concentration was determined using an extinction coefficient of 91mM-1 cm-1 for ∆ε 450-490 nm for ferrous cyt P450-CO complex.24 The oxidized CPR concentration was determined based on the flavin content using an extinction coefficient of 21.4 mM-1cm-1 at 454 nm.25 The concentration of oxidized purified cyt b5 was measured using ε413=117 mM-1 cm-1.26

Steady-state Kinetic Solvent Isotope Effect Enzyme Assays Steady–state kinetic assays of benzphetamine metabolism to formaldehyde were performed under Vmax conditions as previously described.17 Product was measured as described by Nash.27 NADPH oxidation was followed at 340 nm using an extinction coefficient of 6.22 mM-1 cm-1. In the D2O experiments the final concentration of D2O was calculated as being 97 %. Proteins were extensively exchanged with 100 mM potassium phosphate buffer containing 10 % v/v perdeuterated glycerol. The substrate, benzphetamine, and DLPC and were also prepared in D2O. Unless otherwise noted, the final concentration of the constituents in the reaction mixture was: P450, P450 reductase and b5, when present, 0.2 µM, 45 µM DLPC, 1 mM benzphetamine and 50 mM potassium phosphate pH/pD 7.4 or 8. The reaction was started with addition of NADPH to a final concentration of 300 µM and quenched after 6 min at 30 °C with 70 % trifluoroacetic acid in H2Oor D2O.

Preparation of the Reduced cyt P450 - cyt P450 Reductase (P450-CPR) and the cyt P450cyt b5 Complexes

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The cyt P450-CPR and cyt P450-cyt b5 complexes with a 1:1 molar ratio of P450 and reductant were prepared in 100 mM potassium phosphate buffer pH7.4 containing 10 % v/v redistilled glycerol, 2 mM DLPC, 1.6 mM benzphetamine and 5-8 µM methyl viologen (as electron mediator) under anaerobic conditions. The 100 % oxygen saturated buffer contained 100 mM potassium phosphate pH 7.4, 10 % v/v glycerol and 1.6 mM benzphetamine. The complexes were stoichiometrically reduced with a standardized solution of dithionite. The P450-CPR complex was reduced with 3 electrons (1 for P450 and 2 for CPR). The P450 –b5 complex was reduced with 2 electron equivalents (1 for P450, 1 for b5). Before mixing with an equal volume of 100 % oxygen saturated buffer containing of 100 mM potassium phosphate pH7.4, 1.6 mM benzphetamine, and 10 % v/v redistilled glycerol, the concentration of the P450-CPR complex was 274 µM whereas the P450- b5 complex concentration was 440 µM. Rapid Freeze Quench Experiment The rapid freeze quench apparatus consists of a System 1000 syringe driving ram (Update Instruments, Madison, WI) with sample mixer, appropriate reaction hose length, and fastfreezing system based on rapidly spinning silver wheels partially suspended in liquid nitrogen were used in the experiments at room temperature. (The experiments at 15°C were quenched in an isopentane bath at -145°C). Two syringes containing equal volumes (one with the reduced complex and the other with oxygenated buffer) were mounted to the driving ram, with mixer and appropriate hose length attached. Following mixing of the reactants at room temperature (2224°C), the reaction was allowed to proceed in the aging tubing of the instrument. Prior to the freezing of each sample the silver wheels were cleaned, dried, and cooled in liquid nitrogen. At a predetermined time the reaction mixture was ejected from the reaction hose onto the liquid nitrogen-cooled silver wheels. The frozen powder was scraped and packed into a 4mm O.D. 8|Page ACS Paragon Plus Environment

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quartz EPR tube under liquid nitrogen. Once packed, samples were stored in liquid nitrogen. EPR spectra for all samples were recorded within 1 hour of sample quench. Calibration of the reaction hoses was performed by monitoring the reaction of metmyoglobin with azide to give the low spin ferric myoglobin EPR species as described by Ballou and Palmer.28 There is a 5 ms delay related to the distance from the aging tubing to the cold silver wheels and to the time it takes the sample to freeze on the silver wheels. A syringe containing the reduced P450-CPR complex (274 µM) and a second syringe containing 100 % oxygen saturated buffer (100 mM potassium phosphate containing 10 % glycerol and 1.6 mM benzphetamine, pH7.4) were mounted onto the Update instrument at room temperature (2224ºC). The glycerol is necessary to prevent conversion of the P450 to the inactive form cyt P420. An equal volume from the two syringes was mixed and rapidly quenched at 9, 200, 300, 400, 550 and 700 ms after mixing. Following mixing oxygen rapidly binds to ferrous P450 and is reduced by the reductant (either reduced CPR or ferrous b5) and proceeds once through the catalytic cycle. EPR spectra were measured at 30K for the detection of the ferric heme hydroperoxo signal as well as both high and low spin ferric heme signal. For experiments with the P450 2B4-b5complex (440 µM), samples were quenched at 6, 9, 25, 50, 75, 100, 200, and 400 ms after mixing with an equal volume of the complex and 100 % oxygen-saturated buffer at room temperature. After packing, the freeze-quenched samples were stored in liquid nitrogen. The EPR spectra were measured at the CalEPR Center at the University of California, Davis. Continuous-wave (CW) X-band spectra were acquired with a Bruker Elexsys-II E500 spectrometer (Bruker, Billerica, MA) under non-saturating conditions using a Super-High Q resonator (ER 4122SHQE). Cryogenic temperatures were achieved and maintained using an Oxford Instruments ESR900 liquid helium cryostat in conjunction with an Oxford Instruments 9|Page ACS Paragon Plus Environment

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ITC503 temperature and gas flow controller. Data processing was performed with Matlab using the EasySpin 4.0 toolbox.29 Typical collection conditions were 9.38 GHz microwave frequency, 1 mW power, 5 G modulation amplitude, at 30K. A Bruker EMX electron spin resonance spectrometer was used for measurement of the samples mixed and quenched at 15°C. Results Detection of a cyt P450 2B4 Hydroperoxo-Intermediate (Fe3+OOH)-- in the Presence of the P450-cyt P450 Reductase Complex at ~22ºC To determine whether the hydroperoxo intermediate of P450 2B4 was stabilized in the presence of CPR during turnover under ambient conditions, we attempted to trap the ordinarily unstable hydroperoxo-species by rapid freeze quenching of a substrate-bound three electron reduced 1:1 molar ratio P450 2B4-CPR complex (ferrous P450 and the two electron reduced CPR) at various times after starting the reaction by mixing the protein complex with oxygen-saturated buffer. The frozen reaction mixtures were then interrogated by EPR. Recall that only the hydroquinone FMN of the two electron reduced CPR is capable of providing a single electron to P450 and that oxyferrous but not ferrous P450 is able to accept an electron. After mixing, oxygen rapidly binds to the ferrous P450 forming oxyferrous P450 which is quickly reduced by CPR.17 The resulting negatively charged peroxo complex is essentially simultaneously protonated by the efficient 3+

distal-pocket proton delivery network forming the hydroperoxo intermediate (Fe OOH)¯ which has been proposed to exist for a longer time in the presence of P450 reductase than in the presence of b5. By rapid freeze quenching the reaction mixture at various times after mixing with oxygen-saturated buffer and examination of the frozen reaction mixture by EPR it has been possible to detect a ferric heme low-spin P450 2B4 hydroperoxo- species (g- values =2.32, 2.16,

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nd) at the time it was expected to be formed based on similar pre-steady-state spectrophotometric and chemical quench experiments in our laboratory (Figure 2).17

3+

Figure 2 demonstrates that a low-spin, six-coordinate, hydroperoxo (Fe OOH)¯ P450 species with g- values (2.32, 2.16, nd) is observed 300 and 400 ms after mixing the reduced P450 2B4CPR complex with oxygen at room temperature. Since the species with g- values of 2.32 and 2.16 was not observed either earlier or after those mixing times, the data were interpreted to 3+

indicate that the Fe OOH¯ is protonated slowly in the presence of P450 reductase under ambient conditions. As expected, in addition to the hydroperoxo species all spectra reveal the presence of low-spin ferric P450 2B4 and a flavin radical at g=2 reflecting the presence of a 11 | P a g e ACS Paragon Plus Environment

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flavin semiquinone. Control spectra of the oxidized complex are also shown in Figure 2. The oxidized spectrum of full length WT P450 2B4-CPR complex shows a hexa-coordinate, low-spin (S=1/2) rhombic signal from heme bound to water with g- values of 2.42, 2.24 and 1.92 (Table 1) and a penta-coordinate high-spin (S=5/2) rhombic signal, consistent with benzphetamine binding, with g- values of 8.04, 3.55, and 1.70 (spectra not shown). A flavin radical at g= ~2 was not observed in the oxidized spectrum indicating the reductase was oxidized. The assignments of the hydroperoxo EPR signals are based on cryoreduction EPR experiments in which the hydroperoxo-intermediates of several heme proteins were generated and trapped at 77 K and characterized (P450 2B4, P450cam, P450scc, heme oxygenase, nitric oxide synthase, chloroperoxidase, horseradish peroxidase, and myoglobin (Table 1) as described.19,20,30,31,32,33,34,35 To our knowledge this is the first report of a microsomal P450 hydroperoxo –intermediate, generated under single turnover conditions at ambient temperature, being trapped and characterized in the presence of its physiological redox partner, P450 reductase. In cryoreduction experiments the protonation of the peroxo intermediate of P450 2B4 occurs at 77 K where only low energy requiring conformational changes in local wells occur while the protonation of the hydroperoxo intermediate does not occur until ~ 185 K where solvent rotation and rotamer jumps occur.36 This suggests the proton delivery mechanism of the second proton to the distal oxygen of the hydroperoxo moiety is energetically more demanding, and requires greater movement in the active site. Detection of the Hydroperoxo-Intermediate of P450 2B4 in the Presence of the cyt P450P450 Reductase Complex at 15ºC The freeze-quench EPR experiments were repeated at 15ºC since our previous single turnover experiments, which had demonstrated that catalysis was slower with CPR, were performed at 12 | P a g e ACS Paragon Plus Environment

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15ºC. The earlier studies revealed that in the presence of CPR product formation occurred with a rate constant of 0.09 s-1 after mixing the reduced 1:1 molar ratio P450 2B4-CPR complex with oxygen-saturated buffer.17 At the lower temperature the intermediate should also be more stable. When a 1:1 molar ratio of the ferrous P450 2B4: 2 electron reduced reductase was mixed with an equal volume of oxygen-saturated buffer at 15ºC and then rapidly frozen and probed by EPR, a hydroperoxo- species with g- values (2.32, 2.16, 1.946) was observed at ~1500 ms (Figure3).

The time of appearance of the hydroperoxo EPR signal coincides within experimental error with that expected based on the kinetics of product formation in the previous parallel chemical quench experiments.5,17,37 In summary, the hydroperoxo- intermediate has been observed at both room temperature and 15°C. At the higher temperature the hydroperoxo species appeared a shorter 13 | P a g e ACS Paragon Plus Environment

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time after mixing consistent with a faster reaction time. Moreover, the intermediate was detected at the time predicted by formation of product in parallel chemical quench experiments.17 It was also observed when the reaction was conducted in D2O at pD 7.4 (data not shown). Factors contributing to a small signal are: 1) the unstable and transient nature of the intermediate, 2) the insolubility of the active full-length membrane proteins and slower diffusion of O2 in 10 % v/v glycerol, which is ~1500 times more viscous than water,38 3) the inability to effectively synchronize the reaction in contrast to select soluble cyt P450 reactions that can be synchronized with peroxy acids to form Compound I,39,40 4) the inefficient coupling, ~50 %, between NADPH consumption and product formation in microsomal P450s in contrast to cyt P450 cam which is 100 % coupled,5,17 and 5) the limited sensitivity of the freeze-quench EPR experiment, which can only detect low µM concentrations of unpaired spins. Factors that lower the signal in a rapid freeze-quench experiment are frozen water vapor that accumulates while running the freezequench experiment and packing the powder sample. Generally the EPR intensity is decreased by an expected ~50 % of the frozen solution intensity.28 What has probably made the observation of this typically very unstable ferric-hydroperoxo intermediate possible is the 1-2 orders of magnitude slower rate of catalysis of the nonspecific, xenobiotic-metabolizing, microsomal cyts P450 compared to the soluble P450 camphor.17,30 In a slower reaction the catalytic machinery need not be so finely tuned. Lack of a Detectable Hydroperoxo Intermediate in the Presence of a P450-cyt b5 Complex To determine whether a hydroperoxo intermediate accumulated in the presence of b5, a control experiment was conducted using ferrous b5 as the second electron donor rather than the reductase.17 A 1:1 molar complex of ferrous P450 2B4 and ferrous b5 was rapidly mixed at room temperature with oxygen saturated and frozen at various time after mixing (Figure 4). 14 | P a g e ACS Paragon Plus Environment

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Control EPR spectra of low-spin (S=1/2) ferric cyt P450 (g-values = 2.42, 2.24, 1.92) and lowspin (S=1/2) ferric b5 (g-values 3.05, 2.21, nd) and the reduced reaction mixture are shown in Figure 4. The g-values for cyt b5 are in agreement with those found in the literature.41-47 A ferric high-spin signal for cyt P450 was observed at g= 8.12 and 3.55 (spectra not shown) in agreement with previous spectra.19 The first spectrum taken 6 ms after mixing shows the appearance of two rhombic ferric EPR signals of b5 and P450 indicating that b5 has already reduced oxyferrous P450 which has rapidly undergone a catalytic cycle in the dead time of the instrument. Note the absence of a hydroperoxo signal at g=2.3 and 2.184 in any of the EPR spectra recorded. This is the expected result based on similar previous pre steady-state spectrophotometric and chemical 15 | P a g e ACS Paragon Plus Environment

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quench experiments in which catalysis was always faster in the presence of b5. It is presumed that catalysis proceeds via a hydroperoxo intermediate which is too unstable to observe in our experiments.5,17 Product (0.8± 0.05 nmole formaldehyde/nmole P450) was observed in the defrosted quenched sample. Kinetic Solvent Effect on Cytochrome P450 Activity in the Presence and Absence of b5 Kinetic Solvent Isotope Effects (KSIE) have been employed as a probe of water mediated proton delivery in numerous enzymatic reactions.30,48-51 The only steps in the catalytic cycle of the highly coupled cyt P450cam which are expected to exhibit a significant solvent isotope effect are the protonation of the ferric peroxo and ferric hydroperoxo species. Upon protonation the hydroperoxo intermediate undergoes barrierless heterolytic cleavage of oxygen to form compound I.49,52 These two sequential protonation catalytic steps are consistent with a KSIE of ~2 observed in P450 camphor. In an effort to better understand the role of proton delivery in P450 2B4 we have examined the KSIE of b5 on the activity of cyt P450 2B4 in the presence of water and deuterium oxide. In the absence of b5 at pH7.4 the KSIE is 3.23 and 2 with b5. The reaction is 45 % and 20 % coupled in H2O and D2O, respectively (Table2). This indicates that 45 % and 20 % of the reducing equivalents from NADPH were utilized for product formation while the remainder of the reducing equivalents (55 % and 80 % respectively) were diverted to side product formation, primarily H2O2 and D2O2. The catalytic cycle (Figure 1) illustrates that donation of protons to the peroxo and hydroperoxo anions are at the crossroads between productive and nonproductive pathways of cyt P450. The decreased coupling (D2O 20 % vs H2O 45 %) in the absence of b5 indicates that product formation is more dependent on protonation than is side product formation and that both pathways are exquisitely sensitive to the nature of hydration in the active site. 16 | P a g e ACS Paragon Plus Environment

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In the presence of b5 at pH 7.4 the KSIE is slightly lower (Table 2) and is coupled to the same extent (~53 %) in H2O and D2O in agreement with previous studies.15,16,17 The similar amount of coupling in H2O and D2O and slightly attenuated KSIE in the presence of b5 is consistent with the ability of b5 to enhance proton delivery for formation of the active oxidizing species of P450 2B4 at the expense of side product formation. Although it has been known for many years that b5 stimulates the lyase activity of the bifunctional enzyme, P450 17A1, it has only recently been recognized that the stimulatory effect of b5 is a result of increased coupling of the lyase reaction.53,54 The KSIE of P450 2B4 with and without b5 was also compared at pH 8 and pD 8. As expected the KSIE was slightly less at pH 8/pD 8 than at 7.4 presumably because the reaction was somewhat less rate limited by protons (Table3);55 however, the absolute rate of NADPH oxidation was similar in both the protic and deuterium solvents. At pD 8, as at pH 7.4, the coupling of NADPH reducing equivalents to product formation was significantly greater in the presence of b5 (47 % vs 27 %) consistent with the productive proton transfer pathway being more dependent on b5 than the non-productive side product formation. To summarize, the KSIE experiments demonstrate that b5 increased the coupling of the activity of P450 2B4 with a KSIE of ~2 at both pH 7.4 and pH 8. In the absence of b5 the reaction is ~20-30 % less coupled in D2O, suggesting that proton delivery is more rate limiting for product formation than it is for side product formation. The data are also consistent with the increased dependence of Compound I formation in the presence of b5 as a result of more facile proton delivery to the catalytic intermediates. Discussion 17 | P a g e ACS Paragon Plus Environment

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The freeze-quench EPR studies described above support our hypothesis that, although catalysis in the presence of cyt b5 and cyt P450 reductase proceeds via the accepted cyt P450 catalytic cycle (Figure 1) with peroxo, hydroperoxo and oxyferryl porphyrin π cation radical intermediates, the ferric-hydroperoxo intermediate is more stable when P450 reductase binds and reduces oxyferrous P450 than when cyt b5 binds and is the electron donor. These results demonstrate that the redox partners of P450 2B4 are acting as allosteric effectors of a microsomal P450. Putidaredoxin is also recognized as an allosteric effector of P450cam.56 These intriguing results lead one to ask: what is the molecular basis of this distinctive behavior of oxyferrous P450 in the presence of its two reductants? The many (20 as of October, 2016) crystal structures of P450 2B4 bound to different substrates and inhibitors demonstrate the extraordinary plasticity of the active site of this drug metabolizing microsomal P450 and its basic proximal surfaces where the redox partners bind. In an amazing wild type P450 2B4 structure (pdb code 1PO5), the gigantic F-G loop from a one molecule binds to the heme of a second molecule via His 226 in the F-G loop. To accommodate the F-G loop the substrate binding site has undergone a tremendous expansion, and drastic structural changes, including rupture of selected hydrogen bonds to the heme propionates, also occurred at the redox partner binding site on the basic proximal surface of the P450. In view of the known plasticity of active site and proximal surface of cyt P450 2B4, it is proposed that binding of the different redox partners on the proximal surface near the heme allosterically alters the structure of the active site proton delivery pathway to the hydroperoxo intermediate on the distal side of the heme. Allostery is currently understood to arise because of the dynamic and statistical nature of protein conformations and their underlying communication networks. Allosteric changes are believed to result from the binding

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Biochemistry

of an effector compound to one of a dynamic protein’s many conformations and then by mass action, pulling the protein conformational ensemble to the bound state.57 One possible way that b5 could induce a more robust proton delivery pathway of the hydroperoxo species in the P450 active site is by altering the conformation of the highly conserved active site acidic residue, Glu301. Conversely, P450 reductase appears to extend the lifetime of the hydroperoxo intermediate by delaying proton delivery and heterolytic cleavage of the O-O bond. The acidic active site residue corresponding to Glu301 in P450 camphor is Asp251. Asp251 has been shown to be necessary for rapid protonation of the peroxo intermediate which accumulates in the Asp251Asn mutant.18,51 In addition, molecular dynamics calculations have suggested that the conserved Asp251 may also be involved in protonation of the P450cam hydroperoxo intermediate via an active site proton delivery network.58 The molecular dynamics calculations on P450 camphor and the presumed similarity of the P450 2B4 active site proton delivery network to that of P450cam prompted our suggestion that Glu301 of P450 2B4 may participate in delivery of the proton to the hydroperoxo species as well as protonation of the peroxo intermediate. There is ample evidence from structures of P450 2B4 for a flexible Glu301 side chain. The active sites of different P450 2B4 structures demonstrate that although the conformation of Glu301 is variable, two conformations predominate. In one conformation, which is observed in a substrate-free P450 2B4 (pdb 4MVR), the acidic side chain points away from the active site where it is close to the surface of the protein. One side chain oxygen often H-bonds to His172 while the second side chain oxygen H-bonds to a water molecule that in turn H-bonds to a second water molecule in the active site. In the second conformation the Glu301 side chain points into the active site where it H-bonds to an inhibitor in the active site (pdb code 1SUO) or a substrate, paroxetine (pdb code 4JLT). In a P450 2B4 with a 19 | P a g e ACS Paragon Plus Environment

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substrate bound in a catalytically productive conformation, the Glu301 side chain is unlikely to directly provide a proton; however, rotation of the dynamic Glu301 side chain into the active site and interaction with the active site waters may establish a functional proton delivery network to the distal oxygen of the hydroperoxo intermediate.58 ACKNOWLEDGEMENT The work was supported by a NIH grant GM 094209 and VA Merit Review to Lucy Waskell, NIH grant GM 104543 to R. David Britt, and NIH grant GM39451 to Stephen W. Ragsdale. We are grateful for discussions with Dr. Yuting Yang.

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Biochemistry

Table 1. g Values of Ferric High Spin, Low Spin, and Hydroperoxo Species of CYT P450 2B4 and Hydroperoxo g Values of the Indicated Heme Proteins Ferric high spin g1, g2, g3

Ferric low spin g1, g2, g3

a

8.04,3.55, 1.70

2.41, 2.24, 1.92

Bnz+CPR

8.12, 3.54, nd

2.42, 2.24, 1.92

2.32, 2.16, nd

This study

Bnz+CPR

8.04, 3.54, nd

2.4, 2.24, 1.92

2.32, 2.15, nd

This study

Bnz+CPR

8.11, 3.55, nd

2.41, 2.23, 1.92

2.32, 2.16, 1.946

This study

8.12, 3.52, 1.68

2.43, 2.24, 1.92

2.32, 2.18, 1.94

19,20

Protein

Substrate

P450 2B4

Bnz

P450 2B4

P450 2B4

P450 2B4

P450 2B4

BHT

b

Hydroperoxo 3+ Fe OOH¯ g1, g2, g3

Reference

This study

2.39, 2.24, 1.97 P450 2B4

P450cam

P450scc-

None

None

2.43, 2.25, 1.91

2.29, 2.16, 1.94(A)

19,20

Camphor

8.0, 4.0, 1.8

2.41, 2.24, 1.961

2.290, 2.166,1.958

18,59

None

None

None

2.34, 2.181. 1.949

33,34

2.366, 2.182, 1.95

cholesterol

gsNOS-arg

None

None

None

2.31, 2.16, nd

60

None

None

None

2.37, 2.187, 1.924

61

None

None

None

2.32, 2.18, 1.94

62

NO Synthase

Heme oxygenase

Horseradish

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peroxidase

CPO

None

None

None

2.28, 2.176, 1.945

62

None

None

None

2.303, 2.18, 1.946

62

Chloroperoxi -dase

β-chain of hemoglobin

a b Bnz = Benzphetamine / BHT = Butylated hydroxy toluene

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Biochemistry

Table 2. Kinetic Solvent – Isotope Effects of b5 on Product Formation (CH2O), NADPH Oxidation, and Coupling Efficiency by CYT P450 2B4 at pH/pD 7.4 and 30°C.

b5

a

Product Formation H2O D2O

b

NADPH Oxidation H2O D2O

b

Coupling % H2O D2O

Product Formation

kH2O/kD2O

-

36.4±2.5

12.6±1.2

74±7.3

53.3±5.4

45

20

3.23

+

50.6±2.6

26.2±3.7

93.7±3.2

48.4±2.6

55

53

2

a

b

Molar ratio P450:CPR:b5 = 1:1:0 or 1:1:1 Rates are nmol (product or NADPH)/min/nmol P450

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Table 3. Kinetic Solvent – Isotope Effects of b5 on Product Formation (CH2O), NADPH Oxidation, and Coupling Efficiency by Cyt P450 2B4 at pH/pD 8 and 30°C.

b5

a

Product Formation H2O D2O

b

NADPH Oxidation H2O D2O

b

Coupling % H2O D2O

Product Formation

kH2O/kD2O

-

34±2.9

15.4±2.8

78±8.1

54.8±7.3

43

28

2.22

+

50±4.4

28.4±3.7

91±6.6

60.5±4

55

47

1.76

a

Molar ratio P450:CPR:b5 = 1:1:0 or 1:1:1

b

Rates are nmol (product or NADPH)/min/nmol P450. Each result is the average of a minimum of 4 determinations.

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Biochemistry

Figure 1. Catalytic cycle of cytochrome P450. Used with permission of Elsevier, License 3830370432237, March 15, 2016.

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Figure 2. Continuous wave X-band EPR spectra demonstrating formation of a ferric hydroperoxo species by a reduced cyt P450 2B4-CPR complex after mixing at room temperature with oxygenated buffer. (A) EPR spectra of samples quenched between 9-700 ms after mixing a three electron reduced 274 µM complex with an equal volume of 100 % oxygen saturated buffer. For comparison a control EPR spectrum of ferric low spin cyt P450 2B4 is also shown. (B) A magnification of the 200 ms EPR spectrum which lacks the ferric hydroperoxo heme signal. (C) Magnification of the EPR spectrum at 400 ms demonstrating a hydroperoxo signal. Instrument parameters were: temperature 30 K, microwave frequency 9.38 GHz, microwave power 1 mW, modulation amplitude 5 G.

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Biochemistry

Figure 3. Continuous-wave X-band EPR spectra of a reduced P450-CPR complex illustrating formation of a hydroperoxo intermediate at 15°C. EPR spectra of samples quenched at 75 and 1500 ms after mixing a solution of a three electron reduced P450-CPR complex at a concentration of 200 µM with an equal volume of 100 % oxygen-saturated buffer illustrating a hydroperoxo signal. Instrument parameters were: temperature 15 K, microwave frequency 9.382 GHz, microwave power 0.516 mW, modulation amplitude10 G.

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Figure 4. Continuous-wave X-band EPR spectra of a two electron reduced P450-b5 complex lacking evidence of a hydroperoxo intermediate after mixing with oxygen saturated buffer. (A) EPR spectra of quenched samples after mixing at room temperature a two electron reduced P450-b5 complex at a concentration of 440 µM with an equal volume of 100 % O2 saturated buffer. Note the absence of a hydroperoxo signal in any of the traces. Control spectra of the oxidized P450-b5 complex, oxidized b5, and the 2 electron reduced P450-b5 complex are shown. (B) and (C) Show an enlarged view of EPR spectra of the samples obtained at 10 and 400 ms after mixing. Instrument parameters were: temperature 30 K, microwave frequency 9.38 GHz, microwave power 1 mW, modulation amplitude 5 G.

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Corresponding Author Lucy Waskell, M.D., Ph.D., University of Michigan Department of Anesthesiology, Mail Stop 151, 2215 Fuller Road, Ann Arbor, MI 48109-0112. [email protected] Author Contributions Naw May Pearl, Jarett Wilcoxen, Sangchoul Im, Ryan Kunz, and Joseph Darty helped design and conduct experiments as well as analyze the data. Lucy Waskell, R. David Britt, and Stephen W. Ragsdale designed experiments and analyzed the data. All authors contributed to writing the manuscript. All authors have given approval to the final version of the manuscript. Funding The work was supported by a NIH grant GM 094209 and VA Merit Review to Lucy Waskell, NIH grant GM 104543 to R. David Britt, and NIH grant GM 39451 to Stephen W. Ragsdale. References 1. Guengerich, F. P. (2005) Human Cytochrome P450 Enzymes. in Cytochrome P450: Structure, Mechanism and Biochemistry (Ortiz de Montellano, P. R., Ed.), 3rd Ed., pp 377-463, Kluwer Academic/Plenum Publishers, New York. 2. Guengerich, F. P. (2015) Human Cytochrome P450 Enzymes. in Cytochrome P450: Structure, Mechanism and Biochemistry (Ortiz de Montellano, P. R., Ed.), 4th Ed., pp 523-785, Springer International Publishers, Switzerland. 3. Guengerich, F. P., and Munro, A. W. (2013) Unusual cytochrome P450 enzymes and reactions. J. Biol. Chem. 288, 17065-17073.

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4. Vergeres, G., Chen, D. Y., Wu, F. F., and Waskell, L. (1993) The function of tyrosine 74 of cytochrome b5, Arch. Biochem. Biophys. 305, 231-241. 5. Zhang, H., Im, S. C., and Waskell, L. (2007) 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, J. Biol. Chem. 282, 29766-29776. 6. Ortiz de Montellano, P.R. (2015) Substrate Oxidation by Cytochrome P450 Enzymes, in Cytochrome P450: Structure, Mechanism, and Biochemistry, (Ortiz de Montellano, P. R., Ed.) 4th ed., pp 111-176, Springer International, New York. 7. Denisov, I. G. and Sligar, S. G.(2015) Activation of Molecular Oxygen in Cytochrome P450, in Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., Ed.) 4th ed., pp 69-110, Springer International, New York. 8. Lewis, D. F., and Hlavica, P. (2000) Interactions between redox partners in various cytochrome P450 systems: functional and structural aspects, Biochim. Biophy. Acta 1460, 353374. 9. Nagano, S., and Poulos, T. (2005) Crystallographic study on the dioxygen complex of wildtype and mutant cytochrome P450cam, J. Biol. Chem. 280, 31659-31663. 10. Schenkman, J. B., and Jansson, I. (2003) The many roles of cytochrome b5, Pharmacol. Ther. 97, 139-152.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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11. Kim, J.J., and Waskell, L. (2015) Electron Transfer Partners, in Cytochrome P450 Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., Ed.) 4th ed, pp 33-68, Springer International Publishers, Switzerland. 12. Guengerich, F. P. (1983) Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome P-450 reductase related to catalysis in reconstituted systems, Biochemistry, 22, 2811-2820. 13. Guengerich, F. P., Ballou, D. P., and Coon, M. J. (1975) Purified liver microsomal cytochrome P-450 electron accepting properties and oxidation-reduction potential, J. Biol. Chem. 250, 7405-7414. 14. Morgan, E.T., and Coon, M. J. (1984) Effects of cytochrome b5 on cytochrome P450catalyzed reactions studies with manganese-substituted cytochrome b5, Drug Metab. Dispos, 12, 358-364. 15. Gruenke, L. D., Konopka, K., Cadieu, M., and Waskell, L. (1995) The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5, J. Biol. Chem. 270, 24707-24718. 16. Im, S. C., and Waskell, L. (2011) The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b5, Arch. Biochem. Biophys. 507, 144-153. 17. Zhang, H., Gruenke, L., Arscott, D., Shen, A., Kasper, C., Harris, D. L., Glavanovich, M., Johnson, R., and Waskell, L. (2003) Determination of the rate of reduction of oxyferrous

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Page 32 of 38

cytochrome P450 2B4 by 5-deazariboflavin adenine dinucleotide T491V cytochrome P450 reductase, Biochemistry 42, 11594-11603. 18. Davydov, R., Makris, T. M., Kofman, V., Werst, D. E., Sligar, S. G., and Hoffman, B. M. (2001) Hydroxyloation of camphor by reduced oxy-cytochrome P450cam: mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes, J. Am. Chem. Soc., 123, 1403-1415. 19. Davydov, R., Razeghifard, R., Im, S.C., Waskell, L., and Hoffman, B. M. (2008) Characterization of the microsomal cytochrome P450 2B4 O2 activation intermediates by cryoreduction and electron paramagnetic resonance, Biochemistry, 47, 9661-9666. 20. Davydov, R., Im, S., Shanmugam, M., Gunderson, W.A., Pearl, NM, Hoffman, B.M., and Waskell, L. (2016) Role of the proximal cysteine hydrogen bonding interaction in cytochrome P450 2B4 studied by cryoreduction, electron paramagnetic resonance, and electron-nuclear double resonance spectroscopy, Biochemistry 55, 869-883. 21. Saribas, A. S., Gruenke, L. D., and Waskell, L. (2001) Overexpression and purification of the membrane-bound cytochrome P450 2B4, Protein Expression Purif. 21, 303-309. 22. Hamdane, D., Xia, C., Im, SC., Zhang, H., Jung-Ja P. Kim, and Waskell, L. (2009) Structure and function of an NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome P450, J. Biol. Chem. 284, 11374-11384. 23. Mulrooney, S. B., and Waskell, L. (2000) High-level expression in escherichia coli and purification of the membrane-bound form of cytochrome b5, Protein Expression Purif. 19, 173178. 32 | P a g e ACS Paragon Plus Environment

Page 33 of 38

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Biochemistry

24. Omura, T., and Sato, R. (1964) The carbon monoxide binding pigments of liver microsomes: II solubilization, purifications and properties, J. Biol. Chem. 239, 2370-2378. 25. Oprian, D. D., and Coon, M. J. (1982) Oxidation-reduction states of FMN and FAD in NADPH-cytochrome P-450 reductase during reduction by NADPH, J. Biol. Chem. 257, 89358944. 26. Strittmatter, P., and Velick, S. F. (1956) The isolation and properties of microsomal cytochrome, J. Biol. Chem. 221, 253-264. 27. Nash, T. (1953) The colorimetric estimation of formaldehyde by means of the hantzsch reaction, Biochemical J. 55, 416-421. 28. Ballou, D. P., and Palmer, G. A. (1974) Practical rapid quenching instrument for the study of reaction mechanisms by electron paramagnetic resonance spectroscopy, Anal. Chem. 46, 12481253. 29. Stoll, S., and Schweiger, A. (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42-55. 30. Makris, T. M., von Koenig, K., Schlichting, I., and Sligar, S. G. (2007) Alteration of P450 distal pocket solvent leads to impaired proton delivery and changes in heme geometry, Biochemistry 46, 14129-14140. 31. Denisov, I. G., Dawson, J. H., Hager, L. P., and Sligar, S. G. (2007) The ferric-hydroperoxo complex of chloroperoxidase, Biochem. and Biophys, Res. Commun. 363, 954-958.

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Page 34 of 38

32. Denisov, I. G., Makris, T. M., and Sligar, S. G. (2001) Cryotrapped reaction intermediates of cytochrome P450 studied by radiolytic reduction with phosphorus-32, J. Biol. Chem. 276, 11648-11652. 33. Davydov, R., Gilep, A. A., Strushkevich, N. V., Usanov, S. A., and Hoffman, B. M. (2012) Compound I is the reactive intermediate in the first monooxygenation step during conversion of cholesterol to pregnenolone by cytochrome P450scc: EPR/ENDOR/cryoreduction/annealing studies, J. Am. Chem. Soc. 134, 17149-17156. 34. Davydov, R., Strushkevich, N., Smil, D., Yantsevich, A., Gilep, A., Usanov, S., and Hoffman, B. (2015) Evidence that compound I is the active species in both the hydroxylase and lyase steps by which P450scc converts cholesterol to pregnenolone: EPR/ENDOR/cryoreduction/annealing studies, Biochemistry 54, 7089-7097. 35. Egawa, T., Yoshioka, S., Takahashi, S., Hori, H., Nagano, S., Shimada, H., Ishimori, K., Morishima, I., Suematsu, M., and Ishimura, Y. (2003) Kinetic and spectroscopic characterization of a hydroperoxy compound in the reaction of native myoglobin with hydrogen peroxide, J. Biol. Chem. 278, 41597–41606. 36. Lewandowski, J. R., Halse, M. E., Blackledge, M., and Emsley, L. (2015) Direct observation of hierarchical protein dynamics, Science 348, 578-581. 37. Zhang, H., Hamdane, D., Im, S. C., and Waskell, L. (2008) Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4, J. Biol. Chem. 283, 5217-5225.

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Biochemistry

38. Segur, J. B., and Oberstar, H. E. (1951) Viscosity of glycerol and its aqueous solutions, Ind. Eng. Chem. 43, 2117-2120. 39. Spolitak, T., Funhoff, E. G., and Ballou, D. P. (2010) Spectroscopic studies of the oxidation of ferric CYP153A6 by peracids: insights into P450 higher oxidation states, Arch. Biochem. Biophys. 493, 184-191. 40. Rittle, J., and Green, M. T. (2010) Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics, Science 330, 933-937. 41. Aono, T., Sakamoto, Y., Miura, M., Takeuchi, F., Hori, H., and Tsubaki, M. (2010) Direct electrochemical analyses of human cytochrome b5 with a mutated heme pocket showed a good correlation between their midpoint and half wave potentials, J. Biomed. Sci. 17, 90. 42. Davis, C. A., Dhawan, I. K., Johnson, M. K., and Barber, M. J. (2002) Heterologous expression of an endogenous rat cytochrome b5/cytochrome b5 reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands, Arch. Biochem. Biophys. 400, 6375. 43. Davydov, R., and Hoffman, B. M. (2008) EPR and ENDOR studies of Fe(II) hemoproteins reduced and oxidized at 77 K, J. Biol. Inorg. Chem. 13, 357-369. 44. Deng, B., Parthasarathy, S., Wang, W. F., Gibney, B. R., Battaile, K. P., Lovell, S., Benson D. R., and Zhu, H. (2010) Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain, J. Biol. Chem. 285, 30181-30191.

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45. Guzov, V. M., Houston, H. L., Murataliev, M. B., Walker, F. A., and Feyereisen, R. (1996) Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b5. J. Biol. Chem. 271, 26637-26645. 46. Lloyd, E., Ferrer, J.C., Funk, W. D., Mauk, M. R., and Mauk, A. G.(1994) Recombinant human erythrocyte cytochrome b5, Biochemistry 33, 11432-11437. 47. Utecht, R. E., and Kurtz, Jr. D. M. (1988) Cytochrome b5 and NADH-cytochrome-b5 reductase from sipunculan erythrocytes; a methemerythrin reduction system from Phascolopsis gouldii. Biochim.Biophys. Acta 953, 164-178. 48. Schowen, R.L. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W.W., O’Leary, M. H., Northrop, D. B., Eds.), pp 64-99, University Park Press, Baltimore. 49. Aikens, J., and Sligar, S. G. (1994) Kinetic solvent isotope effects during oxygen activation by cytochrome P450cam, J. Am. Chem. Soc. 116, 1143-1144. 50. Vidakovich, M., Sligar, S. G., Li, H., and Poulos, T. L. (1998) Understanding the role of the essential Asp251 in cytochrome P450cam using site-directed mutagenesis, crystallography, and kinetic solvent isotope effect, Biochemistry 37, 9211-9219. 51. Benson, D. E., Suslick, K. S., and Sligar, S. G. (1997) Reduced oxy intermediate observed in D251N cytochrome P450cam, Biochemistry 36, 5104-5107. 52. Kim, S. H., Yang, T. C., Perera, R., Jin, S., Bryson, T. A., Sono, M., Davydov, R., Dawson, J. H., and Hoffman, B. M. (2005) Cryoreduction EPR and 13C, 19F ENDOR study of substrate-

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Page 37 of 38

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Biochemistry

bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant, Dalton Trans. 24, 3464-3469. 53. Peng, H. M., Im, S. C., Pearl, N. M., Turcu, A. F., Rege, J., Waskell, L., and Auchus, R. J. (2016) Cytochrome b5 activates the 17,20-lyase activity of human cytochrome P450 17A1 by increasing the coupling of NADPH consumption to androgen production, Biochemistry 55, 43564365. 54. Akhtar, M., Wright, J. N., and Lee-Robichaud, P. (2011) A review of mechanistic studies on aromatase (CYP19) and 17alpha-hydroxylase-17,20-lyase (CYP17), J. Steroid Biochem. Mol. Biol. 125, 2-12. 55. Swinney, D. C., and Mak, A. Y. (1994) Androgen formation by cytochrome P450 CYP17. Solvent isotope effect and pL studies suggest a role for protons in the regulation of oxene versus peroxide chemistry, Biochemistry 33, 2185-2190. 56. Tripathi, S., Li, H., and Poulos, T. L. (2013) Structural basis for effector control and redox partner recognition in cytochrome P450, Science 340, 1227-1230. 57. Motlagh, H. N., Wrabl, J. O., Li, J., and Hilser, V. J. (2014) The ensemble nature of allostery, Nature 508, 331-339. 58. Taraphder, S., and Hummer, G. (2003) Protein side-chain motion and hydration in protontransfer pathways. Results for cytochrome P450cam, J. Am. Chem. Soc. 125, 3931-3940.

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Page 38 of 38

59. Tsai, R., Yu, C.A., Gunsalus, I.C., Peisach, J., t W. Blumberg, T.W., Orme-Johnson, W.H., and Beinert, H.(1970) Spin-state changes in cytochrome p-45ocam on binding of specific substrates, Proc. Natl. Acad. Sci. U.S.A. 66, 1157-1163. 60. Davydov, R., Sudhamsu, J., Lees, N. S., Crane, B. R. and Hoffman, B. M. (2009) EPR and ENDOR characterization of the reactive intermediates in the generation of NO by cryoreduced oxy-nitric oxide synthase from Geobacillus stearothermophilus, J. Am. Chem. Soc. 131, 1449314507. 61. Davydov, R., Kofman, V., Fujii, H., Yoshida, T., Ikeda-Saito, M., and Hoffman, B. M. (2002) Catalytic mechanism of heme oxygenase through epr and endor of cryoreduced oxy-heme oxygenase and its asp140 mutants, J. Am. Chem. Soc. 124, 1798-1808. 62. Davydov, R., Laryukhin M., Ledbetter-Rogers A., Sono M., Dawson J., and Hoffman BM (2014) Electron paramagnetic resonance and electron-nuclear double resonance studies of the reactions of cryogenerated hydroperoxoferric-hemoprotein intermediates, Biochemistry 53, 4894-4903. Insert Table of Contents Graphic and Synopsis Here

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