STRUCTURAL STUDIES OF IMIDAZOLE-CYTOCHROME-C - RESONANCE ASSIGNMENTS AND STRUCTURAL COMPARISON WITH CYTOCHROME-C

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was u sed to obtain extensive proton resonance assignments of Im-cyt c compl ex which is a possible analog of a late folding intermediate of cytoch rome c. Assignments were made nearly completely for the main-chain and the side-chain protons (all except Gly29). As starting points for the assignment of the Im-cyt c, a limited set of protons was initially as signed by use of 2D NMR magnetization transfer methods to correlated r esonances in the Im-cyt c with assigned resonances in the native cyt c . The subsequent search focused on recognition of main-chain NOE conne ctivity patterns, with use of previously assigned residues to place NO E-connected segments within the amino acid sequence. The observed patt erns of main-chain NOEs provided some structural information and sugge sted potentially significant differences between Im-cyt c and the nati ve cyt c. Differences in NOEs involving side-chain protons were report ed and analyzed. There was evidence for conformational changes induced by the breakage of Fe-S bond. It was concluded that the Im-cyt c had undergone a rearrangement of several regions forming the heme pocket o f the protein. The structural understanding of these effects of the mu tation may be essential to elucidate the changes in function and kinet ic mechanism of cyt c folding.