Factors affecting the heme electronic structure of proteins with heme c

Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2012.Cytochromes c (cyts c) are excellent model systems for the detailed study of factors that influence heme reactivity. Cyts c are electron-transfer proteins containing iron heme covalently attached to a Cys-X-X-Cys-His (CXXCH) motif. His in this motif axially coordinates the heme iron, and the other axial ligand in the proteins of this study is Met. In this study, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and resonance Raman (rR) spectroscopic techniques as well as X-ray crystallography are being used to understand the effects of heme distortion, axial bond strength, and second-sphere interactions on heme electronic structure. We studied a series of mutants of cytochromes c from Pseudomonas aeruginosa (Pa c-551) and from Hydrogenobacter thermophilus (Ht c-552) and found that mutations introduced near or in the CXXCH motif result in changes in heme molecular and electronic structure. In Pa c-551, mutation of a Phe interacting with the CXXCH motif to Ala is known to enhance heme ruffling. Analysis of ligand field parameters obtained from EPR spectra reveal a clear relationship between heme ruffling and energies of the dxy, dxz, and dyz orbitals. In Ht c-552, mutations previously shown to alter the His-Fe(III) bond strength are demonstrated also to change heme ruffling. Similarly, heme conformation and axial bond strength are shown to alter the electronic structure and the spin state of the heme proteins with an open coordination site by using Ht c-552 variants lacking the axial Met ligand. The N64Δ mutation of Nitrosomonas europaea cyt c552 (Ne c-552) causes significant changes in the electronic structure. Structural causes of the changes of the electronic properties upon this mutation were investigated by X-ray crystallography on the wild-type and N64Δ variant of Ne c-552. We studied the axial Met ligation loss in Ht c-552 when adsorbed onto pyrolytic graphite edge (PGE) electrodes during protein film voltammetry (PFV) using Ht c-552, Ht M61A, and Ht M61H. We showed that the required driving force for reorganization of the axial Met bearing loop of Ht c-552 is lowered by the PGE surface