Oxidation and electronic state dependence of proton transfer in the enzymatic cycle of cytochrome P450eryF.
Hydrogen bond networks, consisting of hydrogen bonded waters anchored by polar/acidic amino acid sidechains, are often present in the vicinity of the oxygen binding clefts of P450s. Density functional and quantum dynamics calculations of a O(2) binding cleft network model of cytochrome P450eryF(CYP107A1) indicate that such structural motifs facilitate ultrafast proton transfer from network waters to the dioxygen of the reduced oxyferrous species via a multiple proton translocation mechanism with barriers of 7-10 kcal/mol on its doublet ground state, and that the energies of the proton transfer reactant and constrained proton transfer products have an electronic and oxidation state dependence [J. Am. Chem. Soc. 124 (2002) 1430]. In the present study, the origin of the oxidation state dependence is shown to have its roots in differential proton affinities while the electronic state dependence of the reduced oxyferrous heme has its origins in subtle differences in network topologies near the transition state of the initial proton transfer event. Relaxed potential surface scans and unconstrained proton transfer product optimizations indicate that the proton transfer product in both the singlet oxyferrous heme and the reduced oxyferrous heme species in a quartet state are not viable stable (bound) states relative to the reactant form. While the proton affinity of H(3)O(+) is sufficient for it to protonate both the oxyferrous and the reduced oxyferrous heme species, hydrogen bond network stabilized water is only capable of protonating the reduced oxyferrous form. This interpretation is substantiated by study of the NO bound reduced ferrous heme of P450nor, which is isoelectronic with the oxyferrous heme and has a similar proton affinity. Density functional calculations on a more extensive O(2) binding cleft model support the multiple proton translocation mechanism of transfer but indicates that the significant negative charge density on the bound dioxygen of the reduced oxyferrous heme species, in its doublet ground state, polarizes the associated hydrogen bond network sufficiently so as to result in short, strong, low-barrier hydrogen bonds. The computed O-H-O bond distances are less than 2.55 A and have a near degeneracy of the proton transfer reactant and initial (sudden) proton transfer products. These low-barrier hydrogen bond features, in addition to the finding of a (zero point uncorrected) barrier of 1.3 kcal/mol, indicate that proton transfer from water to the distal oxygen should be rapid, facile and may not require large curvature tunneling as originally suggested by use of a smaller model. An initial assessment of protonation of the reduced oxyferrous heme distal oxygen by a model of 6-deoxyerythronolide B (6-DEB) indicates it to be low barrier (3.8 kcal/mol) and exothermic (-2.9 kcal/mol). The combined results indicate the plausibility of simultaneous diprotonation of the distal oxygen of the reduced oxyferrous heme, leading to O-O bond scission, using the combined water network and 6-DEB substrate protonation agents.
Harris
Danni L
DL
Molecular Research Institute, Mountain View, CA 94043, USA. dannil@purisima.molres.org
D001426:Bacterial Proteins; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011199:Potentiometry; D011487:Protein Conformation
D001426:Bacterial Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea; D010100:Oxygen