Loss of Either of the Two Heme-binding Cysteines from a Class I c-Type Cytochrome Has a Surprisingly Small Effect on Physicochemical Properties

Abstract

Almost without exception, c-type cytochromes have heme covalently attached via two thioether linkages to the cysteine residues of a CXXCH motif. The reasons for the covalent attachment are not understood. Reported here is cytoplasmic expression in Escherichia coli of AXXCH and CXXAH variants of cytochrome c 552 from Hydrogenobacter thermophilus; remarkably, the single thioether bond proteins have, apart from an altered visible absorption spectrum, almost identical properties, including thermal stability and reduction potential, to the wild type CXXCH protein. In combination with previous work showing that an AXXAH variant of cytochrome c 552 is much less stable than the CXXCH form, it can be concluded that covalent attachment of heme via either of thioether bonds is sufficient to confer considerable stability and that these bonds contribute little to the setting of the reduction potential. The absence of AXXCH or CXXAH heme-binding motifs from bacterial cytochromes c may relate to the coexistence of the assembly pathway with that for formation of disulfide bonds in the bacterial periplasm.

Heme Is Not Required forAquifex aeolicusCytochromec555Polypeptide Folding

“…The large difference in T m value between holo AA c 555 and holo C12A/C15A, about 50 C, indicates that thioether bonds greatly increase the stability of the overall protein structure, consistently with previous findings for other cytochromes. 15,16,21) Although the T m value of the apo C12A/C15A variant was only about 5 C lower than that of the holo C12A/ C15A variant, cooperativity during protein denaturation was reduced in the apo protein (Fig. 2), suggesting that the heme affects the denaturation process in C12A/ C15A, but the thermal denaturation of apo C12A/C15A still exhibited cooperativity comparable to that of its holo form (Fig.…”

In Vitro Studies on Thioether Bond Formation between Hydrogenobacter thermophilus Apocytochrome c552 with Metalloprotoporphyrin Derivatives

“…Wild-type, C11A, C14A, and C11A/C14A variants of Hydrogenobacter thermophilus c 552 were expressed and purified as previously described (9,10). Apocytochromes c were prepared analogously to the method of Fisher et al (11).…”

C-type cytochromes: diverse structures and biogenesis systems pose evolutionary problems

“…As described above, H. thermophilus cytochrome c 552 , which has a typical CXXCH haem-binding motif, and mutants with AXXCH and CXXAH motifs can, exceptionally, be expressed as stable holocytochromes in the cytoplasm of E. coli (see § 2a; Tomlinson & Ferguson 2000b). By targeting these proteins to the periplasm of E. coli using the signal peptide of a bacterial cytochrome c we have assessed the ability of the Ccm system to attach haem covalently to proteins with one or two cysteine residues in the haem-binding motif.…”

“…with AXXCH or CXXAH haem-binding motifs) could be isolated and their physico-chemical properties investigated (Tomlinson & Ferguson 2000b). For each of these proteins, the haem is covalently attached to the polypeptide through a single thioether bond.…”