Kelath Murali Manoj

We sought to clarify on the hitherto unresolved role of N-terminal transmembrane segments (TMS) of cytochrome P450 (CYP) and its’ reductase (CPR) in protein interaction/catalysis. TMS analyses show little evolutionary conservation in CYPs. The conserved CPR’s TMS poses limited scope for predictable/consistent hetero-recognition with the wide bevy of CYPs’ TMS, as evident from preliminary analyses and TMhit server predictions for inter-helical binding. Further, experimentations with four different CPR preparations (preps) and two liver microsomal CYPs (2C9 and 2E1) shows that the hydroxylated product formation rate is not quantitatively correlated to the extent of integrity of the CPR N-terms. Incorporation of cytochrome b ₅ in some reactions afforded similar rates while employing either fully intact or partially intact CPR. A survey of literature shows that liver microsomal CYPs function quite well even without the TMS or with significantly altered TMS. These observations negate the hypothesis that N-term TMS of CPR or CYP is obligatory for CYP–CPR interaction and catalysis. Also, in CYP2E1-mediated hydroxylation of para-nitrophenol, the extent of intactness or truncation did not significantly affect the CPR preps’ catalytic role at very low or high substrate concentrations. To interpret these results, we draw support from recently published research on reduced nicotinamide adenide dinucleotide phosphate oxidase (Takac et al., J Biol Chem, 286:13304–13313, 2011) and from our pertinent earlier works. We infer that CPR’ free TMS segment could alter the diffusible reactive oxygen species’ dynamics in the microenvironment, thereby altering the reaction outcome. Based on the evidence, we conclude that TMS merely facilitates “interaction/catalysis” by anchoring the CYP and CPR in the lipid interface.

The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1∶1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100∶1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the amount of substrate metabolized. We aimed to find the rationale to explain for these two oddities. We report here that CPR is capable of activating molecular oxygen on its own merit, generating diffusible reduced oxygen species (DROS). Also, in the first instance for a flavoprotein, CPR is shown to deplete peroxide via diffusible radical mediated process, thereby leading to the formation of water (but without significant evolution of oxygen). We also quantitatively demonstrate that the rate of oxygen activation and peroxide depletion by CPR accounts for the major reactivity in the CYP+CPR mixture. We show unambiguously that CPR is able to regulate the concentration of diffusible reduced oxygen species in the reaction milieu. These findings point out that CPR mediated processes are bound to be energetically ‘wasteful’ and potentially ‘hazardous’ owing to the unavoidable nature of the CPR to generate and deplete DROS. Hence, we can understand that CPR is distributed at low densities in cells. Some of the activities that were primarily attributed to the heme-center of CYP are now established to be a facet of the flavins of CPR. The current approach of modeling drugs to minimize “uncoupling” on the basis of erstwhile hypothesis stands questionable, considering the ideas brought forth in this work.

Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO) characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP), uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation) of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model) was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the “steady state kinetics” of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates) is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s) affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of “the product of interest” in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of “substrate concentration at maximum observed rate”. The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes.