Tuning the Reduction Potential of Engineered Cytochrome c-553

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Abstract
Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low reduction potential with respect to other c-type cytochromes. Solvent heme exposure has been indicated as one of the most important factors in modulating the midpoint potential of the redox center. To test this hypothesis, a unique surface-exposed cysteine has been substituted for either M23 or G51 to produce the corresponding mutants and allow the formation of homodimers through a specific disulfide bridge. The reduction potentials, determined via spectroelectrochemistry, show an increase from +20 ± 5 mV for the wt to +88 ± 5 and +105 ± 5 mV for the M23C−M23C homodimer and G51C−G51C homodimer, respectively. Chemical denaturation of the homodimers leads to parameters related to the hydrophobicity (m) and the number of buried side chains (nB), which suggest a decrease of exposure of the heme as a result of dimerization. These results are consistent with the heme-accessible surface area (ASA) calculated from a computer model of the homodimers. The ASA values show a decrease from 73 Å2 for the wt to 66 and 50 Å2 per heme for the M23C−M23C homodimer and G51C−G51C homodimer, respectively. The trend of the m- and nB-values, the degree of solvent accessibility, and the midpoint potential observed upon formation of the homodimers indicate a correlation between the reduction potential values and the exclusion of water from the heme surface.