Factors that determine the unusually low reduction potential of cytochromec(550) in cyanobacterial photosystem II

A new purification protocol for cytochrome c(550) (cyt c(550)) from His-tag ged Synechocystis PCC 6803 photosystem II (PSII) was developed which allows the protein to be isolated in high yield and purity. Electron paramagnetic resonance spectroscopy of cyt c(550), both free in solution and in intact PSII preparations, yields identical spectra with g values at 1.50, 2.23, an d 2.87, which are characteristic for a ferric low-spin bis-histidine coordi nated heme. The resonance Raman spectrum of the isolated protein exhibits f eatures characteristic of bis-histidine axial ligation of the iron and a sl ight ruffling of the heme macrocycle. To-ether, these results indicate that the heme structure is not very different from most c-type cytochromes, and thus the structure of the heme does not account for its unusually low redu ction potential. A direct electrochemical measurement of the reduction pote ntial was performed using square,wave voltammetry on a pyrolytic graphite e dge electrode, yielding E-1/2= -108 mV (vs. NHE) with a peak separation of 5 mV. This value is 150 mV more positive than that previously measured by r edox titrations. Because the behavior of the protein in the electrochemistr y experiments is indicative of adsorption to the electrode surface, we surm ise that binding of the protein to the electrode excludes solvent water fro m the heme-binding site. We conclude that the degree of solvent exposure ma kes a significant contribution to the heme reduction potential. Similarly, the binding of cyt c(550) to PSII may also reduce the solvent exposure of t he heme, and so the direct electrochemical value of the reduction potential may be relevant to the protein in its native state.