Modulation of Ligand-Field Parameters by Heme Ruffling in Cytochromes <i>c</i> Revealed by EPR Spectroscopy

Electron paramagnetic resonance (EPR) spectra of variants of <i>Hydrogenobacter thermophilus</i> cytochrome <i>c</i>₅₅₂ (<i>Ht c</i>-552) and <i>Pseudomonas aeruginosa</i> cytochrome <i>c</i>₅₅₁ (<i>Pa c</i>-551) are analyzed to determine the effect of heme ruffling on ligand-field parameters. Mutations introduced at positions 13 and 22 in <i>Ht c</i>-552 were previously demonstrated to influence hydrogen bonding in the proximal heme pocket and to tune reduction potential (<i>E</i>_m) over a range of 80 mV [Michel, L. V.; Ye, T.; Bowman, S. E. J.; Levin, B. D.; Hahn, M. A.; Russell, B. S.; Elliott, S. J.; Bren, K. L. <i>Biochemistry</i> <b>2007</b>, <i>46</i>, 11753–11760]. These mutations are shown here to also increase heme ruffling as <i>E</i>_m decreases. The primary effect on electronic structure of increasing heme ruffling is found to be a decrease in the axial ligand-field term Δ/λ, which is proposed to arise from an increase in the energy of the d_<i>xy</i> orbital. Mutations at position 7, previously demonstrated to influence heme ruffling in <i>Pa c</i>-551 and <i>Ht c</i>-552, are utilized to test this correlation between molecular and electronic structure. In conclusion, the structure of the proximal heme pocket of cytochromes <i>c</i> is shown to play a role in determining heme conformation and electronic structure