Previously, in vitro formation of thioether bonds between Hydrogenobacter thermophilus apocytochrome c552 and Fe-protoporphyrin IX has been demonstrated (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc Natl Acad Sci U S A 99, 7872-6). Now we report studies on the reaction between the metalloderivatives Zn-, Co-, and Mn-protoporphyrin IX with the cysteine thiols of Hydrogenobacter thermophilus apocytochrome c552. All these metalloporphyrins were capable of forming a “b-type cytochrome” state in which the hydrophobic prosthetic group is bound non-covalently. Zn(II)-protoporphyrin IX attached to the polypeptide covalently in the presence of either dithiothreitol or tri(2-carboxyethyl)phosphine to keep the thiol moieties reduced. These data show that the chemical nature of the thiol reducing agent does not interfere with the thioether bond forming mechanism. Mn-porphyrin could only react with the protein in the divalent state of the metal ion. Co-porphyrin did not react with the cysteine thiols of the apocytochrome in either oxidation state of the metal. In the absence of a metal (i.e. protoporphyrin IX itself) no reactivity toward apocytochrome is observed. These results have significant implications for the chemical requirements for thioether bond formation of heme-vinyl groups to cysteine thiols and also have potential applications in de novo design of metalloproteins.