Molecular modeling of cytochrome b₅ with a single cytochrome c-like thioether linkage

Bovine liver cytochrome b (5) (cyt b (5)), with heme bound noncovalently, has been converted into a cyt c-like protein (cyt b (5) N57C) by constructing a thioether linkage between the heme and the engineered cysteine residue. With no X-ray or NMR structure available, we herein performed a molecular modeling study of cyt b (5) N57C. On the other hand, using amino acid sequence information for a newly discovered member of the cyt b (5) family, domestic silkworm cyt b (5) (DS cyt b (5)), we predicted the protein structure by homology modeling in combination with MD simulation. The modeling structure shows that both Cys57 in cyt b (5) N57C, and Cys56, a naturally occurring cysteine in DS cyt b (5), have suitable orientations to form a thioether bond with the heme 4-vinyl group, as the heme is in orientation A. In addition to providing structural information that was not previously obtained experimentally, these modeling studies provide insight into the formation of cyt c-like thioether linkages in cytochromes, and suggest that c-type cyt b (5) maturation involves a b-type intermediate.