INFLUENCE OF CHARGE AND POLARITY ON THE REDOX POTENTIALS OF HIGH-POTENTIAL IRON-SULFUR PROTEINS - EVIDENCE FOR THE EXISTENCE OF 2 GROUPS

We have investigated the HiPIPs from Ectothiorhodospira vacuolata (iso -l and iso-2), Chromatium vinosum, Rhodocyclus gelatinosus, Rhodocyclu s tenuis (strain 2761), Rhodopila globiformis, and Rhodospirillum sali narum (iso-2) by direct electrochemistry. Using a glassy carbon electr ode with a negatively charged surface, direct, unpromoted electrochemi stry is possible with the positively charged HiPIPs. With the negative ly charged HiPIPs, the positively charged and flexible bridging promot er poly (L-lysine) is required. The stability of the response can be i mproved by morpholin, aspartate, tryptophan, or 4,4'-dipyridyl. These ''stabilizers'' prevent the blocking of the electrode by denatured pro tein. The redox potential of 500 mV found for R. salinarum iso-2 is th e highest HiPIP potential reported. The presence of histidines in the sequence does not per se predict a pH-dependent redox potential. Only C. I vinosum and R. gelatinosus HiPIPs show a weak but significant pH dependence with a difference of 35 mV between the low- and the high-pH form and maximum slopes of -20 mV/unit. The dependence of the midpoin t potential on temperature and on ionic strength varies over the diffe rent HiPIPs. The dependence of the potentials on root I cannot be full y explained by the Debye-Huckel theory because the linearity exceeds t he limiting concentration and only small negative slopes are observed (0 to -28 mV/root M). Combination of the sequences, the optical spectr a, the overall charges, and the redox thermodynamics suggests the exis tence of two groups of HiPIPs. One group consists of Chromatium-like H iPIPs with redox potentials between 300 and 350 mV, modulated only by the solvation of the cluster. The second group is formed by Ectothiorh odospira-like HiPIPs with potentials between 50 and 500 mV, modulated by the overall charge of the Peptide (25 mV/unit) and by the solvation of the cluster.