Abstract
The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization.
Highlights
Cytochrome P450s (CYPs) form a ubiquitous enzyme family which is directly involved in the oxidation of a wide range of organic compounds including drugs, steroids, and vitamins.[1]
The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels
Such a dehydration of the catalytic site is essential for starting the enzymatic cycle of cytochrome P450 proteins,[17] since this favors the reduction of the ferric species 2 by cyt P450 reductase (CPR, first Electron Transfer, ET) due to increase of the Fe(II)/Fe(III) redox equilibrium towards the ferrous counterpart 3.[18,19,20]
Summary
Cytochrome P450s (CYPs) form a ubiquitous enzyme family which is directly involved in the oxidation of a wide range of organic compounds including drugs, steroids, and vitamins.[1]. O2 binds to the heme iron (4), which undergoes a second reduction (5) and a protonation to form the Fe(III)OOH- intermediate 6, called Cpd 0 The latter undergoes in turn, a second protonation on the distal OH, resulting in heterolytic O-OH bond cleavage, and generation of the iron-oxo porphyrin-cation radical intermediate, Por+ Fe(IV) = O 7, so called Cpd I.[21,22,23,24,25,26] This Por+ Fe(IV) = O species, which is the ultimate oxidant of the enzyme, catalyzes the efficient mono-oxygenation of many different substrates, e.g., 8.[14,24,27,28,29,30,31,32,33] Alternatively, the protonation of the proximal oxygen atom of the Cpd 0 species is responsible for the release of hydrogen peroxide (H2O2), thereby uncoupling the oxygen consumption and substrate oxidation (see central arrow in Fig 1).[34,35,36] The overall coupled process may be summarized as follows: RH þ O2 þ 2eÀ þ 2Hþ !
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