Experimental evidence for the role of buried polar groups in determining the reduction potential of metalloproteins: the S79P variant of Chromatium vinosum HiPIP

The amide group between residues 78 and 79 of Chromatium vinosum high-poten tial iron-sulfur protein (HiPIP) is in close proximity to the Fe4S4 cluster of this protein and interacts via a hydrogen bond with S gamma of Cys77, o ne of the cluster ligands, The reduction potential of the S79P variant was 104+/-3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distributio n in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is d ue to the different electrostatic properties of amide 79 in these two prote ins, These results suggest that the influence of amide group 79 on the redu ction potential of C, vinosum HiPIP is a manifestation of a general electro static effect rather than a specific interaction. More generally, these res ults provide experimental evidence for the importance of buried polar group s in determining the reduction potentials of metalloproteins.