Assignment of heme resonances in the 1H NMR spectrum of oxidized Desulfovibrio vulgaris (Hildenborough) cytochrome c3

Abstract

A combination of 2D COSY and NOESY experiments and 1D nuclear Overhauser effect measurements have allowed an extensive assignment of the hyperfine-shifted heme resonances in the downfield region of the 1H NMR spectrum of the fully oxidized tetraheme cytochrome c 3 from Desulfovibrio vulgaris, Hildenborough. Fifteen of the sixteen heme methyl groups were identified and assigned, some on a tentative basis. Most of the one-proton paramagnetic resonances were demonstrated to arise from the α-CH 2 groups of the heme propionate side chains, and from the β-CH 2 groups of the iron-binding axial histidines. Assignment of methyl resonances to individual hemes was achieved by reference to previous calculations of electron density distributions on the four hemes of the highly homologous Miyazaki protein obtained by NMR data ( Biochemistry, 29 (1990) 2257). The methyl hyperfine shift patterns are apparently different for the individual hemes. Despite the complexity of the system due to interheme interactions, we have determined that these patterns appear to be influenced by the orientation of the imidazole planes of the iron-binding axial histidines, as found for simpler monoheme proteins.