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J. Biol. Chem., Vol. 279, Issue 44, 45347-45353, October 29, 2004

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

Oliver Daltrop and Stuart J. Ferguson

From the Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom

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, 7872–7876). Now we report studies on the reaction between the metalloderivatives Zn-, Co-, and Mn-protoporphyrin IX and the cysteine thiols of H. thermophilus apocytochrome c₅₅₂. All of these metalloporphyrins were capable of forminga"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.

Received for publication, July 29, 2004 , and in revised form, August 17, 2004.

^* This work was supported by Grants C20071 from the UK Biotechnology and Biological Sciences Research Council (to S. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A Junior Research Fellow of Christ Church, Oxford. To whom correspondence may be addressed. Tel.: 44-1865-275240; Fax: 44-1865-275259; E-mail: oliver.daltrop{at}bioch.ox.ac.uk. The W. R. Miller fellow of St. Edmund Hall. To whom correspondence may be addressed. Tel.: 44-1865-275240; Fax: 44-1865-275259; E-mail: stuart.ferguson{at}bioch.ox.ac.uk.

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