INVESTIGATION OF THE PROTON-ASSISTED PATHWAY TO FORMATION OF THE CATALYTICALLY ACTIVE, FERRYL SPECIES OF P450S BY MOLECULAR-DYNAMICS STUDIES OF P450ERYF

The recently determined crystal structure of cytochrome P450eryF (6-de oxyerythronolide B hydroxylase; CYP107Al) in its ferric heme substrate -bound form has been used to address one of the most fundamental unres olved aspects of the mechanism of oxidation common to this ubiquitous family of metabolizing heme proteins, the pathway from the twice reduc ed dioxygen species to the putative catalytically active ferryl oxygen species. Both of these species are too transient to have been charact erized experimentally, and the transformation from one to the other ha s been only partially characterized. The observed requirement of two p rotons and the formation of water in this transformation suggests a pr oton-assisted dioxygen bond cleavage as a plausible pathway. However, this pathway is difficult to establish by experiment alone, and the so urce of the protons in the largely hydrophobic binding pocket of the P 350s remains unclear. In this work we have performed molecular dynamic s simulations of the twice reduced dioxygen substrate-bound form of th is isozyme in order to (i)determine the plausibility of the proposed p athway to compound I formation, a proton-assisted cleavage of the diox ygen bond, and (ii) investigate the possible source of these protons. The analysis of the molecular dynamics trajectories of this species do es indeed provide further evidence for this pathway and points to a so urce of protons. Specifically, two dynamically stable hydrogen bonds t o the distal oxygen atom of the dioxygen ligand, one by the substrate and the other by a bound water, are found, consistent with the propose d proton-assisted cleavage of the bond and formation of water. In addi tion, an extensive dynamically stable hydrogen bond network is formed that connects the distal oxygen to Glu 360, a well-conserved residue i n a channel accessible to solvent that could be the ultimate source of protons. The simulations were done for both a protonated and unproton ated Glu and led to a proposed mechanism of proton transfer by it to t he distal oxygen atom. In order to validate the procedures used for th e simulation of this transient twice-reduced species, we have used the se same procedures to perform molecular dynamics simulations of two ot her forms of P450eryF, the ferric and ferryl substrate-bound species, and compared the results with experiment. The results for the ferric s ubstrate-bound species were assessed by comparisons to the experimenta lly determined X-ray structure and fluctuations, and good agreement wa s found. The simulations performed for the ferryl substrate-bound spec ies led to the correct prediction of the observed regio- and stereospe cific hydroxylation of its natural substrate, 6-deoxyerythronolide B ( 6-DEB) at the 6S position. The results of these two additional studies lend credibility to the important mechanistic inferences from the sim ulations of the transient twice reduced dioxygen species: further evid ence for a proton-assisted pathway from it to the catalytically active ferryl species and a possible source of the protons.