Development of spectroscopic probes of second-sphere interactions in cytochromes c

Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2010.Heme reactivity is central to a wide range of biological functions, including gas transport, catalysis and electron transfer. Nature employs an array of methods by which reactivity is modulated in heme containing proteins. In this thesis, spectroscopic methods are developed and used to elucidate details of how reduction potential is tuned by the protein environment in and around the active site of cytochromes c. Variants from two homologous bacterial cytochromes c, Pseudomonas aeruginosa cytochrome c551 and Hydrogenobacter thermophilus cytochrome c552, are examined to gain insight into factors that contribute to modulation of redox potential in these electron transfer proteins. Heteronuclear NMR techniques are used to directly probe nuclei in the axial His ligand. NMR chemical shifts and vibrational frequencies of exogenous ligands bound trans to the axial His are also investigated. Of particular interest is how second-sphere mutations alter histidinate character of the axial His, and the impact of these changes in histidinate character on reduction potential. The results indicate that as histidinate character increases, reduction potential decreases. Distortions of the heme cofactor from planarity are also hypothesized to be a factor in tuning redox potential. The effect of a ruffling distortion on redox potential in two variants from Pseudomonas aeruginosa cytochrome c551 are examined using heteronuclear NMR techniques. Results indicate that increasing the ruffling distortion corresponds to a decrease in redox potential. These results show how NMR techniques can be used to probe subtle interactions in heme-containing biological systems. In addition to investigating factors that impact redox potential in cytochromes c, this thesis work also presents results using Nuclear Resonance Vibrational Spectroscopy (NRVS). NRVS is a synchrotron-based vibrational technique that is uniquely suited to directly measure vibrations involving iron. NRVS is an important development that gives an additional tool for investigation of heme containing proteins. The novel techniques developed in this project have profound implications for our ability to observe the active sites in heme proteins