Assignment of hyperfine-shifted resonances in yeast ferricytochrome c isozyme 2 using the proton pre-steady-state nuclear Overhauser effect.

Yeast cytochrome c isozyme 2 is one of two cytochrome c isozymes which are found in yeast mitochondria. Unlike isozyme 1, which can dimerize in vitro due to a free sulfhydryl group at primary sequence position 102, isozyme 2 (Ala-102) is a monomer. The hyperfine proton NMR resonance pattern of ferric isozyme 2 is somewhat different from the horse and tuna ferricytochromes c. Thus, resonance assignments would help determine how similar the yeast, horse and tuna proteins actually are. In this work, many of the unassigned proton hyperfine resonances of the ferric protein have been assigned using the proton pre-steady-state nuclear Overhauser effect. Assigned resonances include those attributable to the heme 7-propionic acid alpha-CH2, His-18 alpha-CH and beta-CH2, Met-80 beta-CH2, and heme 4-beta-CH3 protons. The overall pattern of NOE connectivities is similar to the horse and tuna proteins. Combining shift and NOE patterns leads to the conclusion that the heme environment of yeast ferric isozyme 2 in solution is similar, but not identical, to the heme environment of horse and tuna cytochromes c.