Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2014.
Heme is a biological cofactor that performs an array of functions, including electron transfer,
redox catalysis, and gas sensing and transport. The ligands and local environment
of the heme group are essential in tuning the properties of the heme to perform in such
diverse roles. A subset of heme cofactors, known as hemes c, are covalently ligated to the
protein backbone via a CXXCH peptide motif, and are mainly dedicated to performing
electron transfer. The research described in this thesis focuses on the ways in which covalent
attachment of heme c tunes heme properties relevant to electron transfer. The heme
attachment motif is known to promote an out-of-plane distortion of the heme called ruffling.
Variants of bacterial cytochromes c from Hydrogenobacter thermophilus and Pseudomonas
aeruginosa in which the magnitude of heme ruffling has been altered were analyzed by their
paramagnetic NMR shifts, enabling a full description of the influence of ruffling on heme
hyperne shifts. The analysis was then used to determine the influence of the length of the
heme attachment motif, which covalently binds the heme, on the extent of heme ruffling.
The analysis determined that in H. thermophilus cytochrome c, both longer (CX4CH) and
shorter (CX1CH) heme attachment motifs enhance the heme ruffling distortion. Increased
heme ruffling, measured by the hyperfine NMR shift analysis, correlates to a decreased
redox potential of the heme in a number of cytochrome c variants, suggesting that a biological
role of the heme attachment motif may be to tune the redox potential of the heme
to lower potentials via the heme ruffling distortion. The vibrational profile of the heme attachment
motif was also investigated with nuclear resonance vibrational spectroscopy, with
implications for understanding how heme covalent attachment optimizes electron transfer
through vibrational coupling. Finally, an application of the covalent attachment of heme
to a peptide derived from cytochrome c is developed via substitution of cobalt for the
heme iron. The resulting cobalt microperoxidase is demonstrated to be a rare example of a
hydrogen-evolving electrocatalyst that functions in neutral water using a non-noble metal.