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J. Biol. Chem., Vol. 278, Issue 52, 52075-52083, December 26, 2003

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A Cytochrome b₅₆₂ Variant with a c-Type Cytochrome CXXCH Heme-binding Motif as a Probe of the Escherichia coli Cytochrome c Maturation System^*

James W. A. Allen, Paul D. Barker¶, and Stuart J. Ferguson||

From the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom and the ^¶Chemistry Department and the Centre for Protein Engineering, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

Cytochrome b₅₆₂ is a periplasmic Escherichia coli protein; previous work has shown that heme can be attached covalently in vivo as a consequence of introduction of one or two cysteines into the heme-binding pocket. A heterogeneous mixture of products was obtained, and it was not established whether the covalent bond formation was catalyzed or spontaneous. Here, we show that coexpression from plasmids of a variant of cytochrome b₅₆₂ containing a CXXCH heme-binding motif with the E. coli cytochrome c maturation (Ccm) proteins results in an essentially homogeneous product that is a correctly matured c-type cytochrome. Formation of the holocytochrome was accompanied by substantial production of its apo form, in which, for the protein as isolated, there is a disulfide bond between the two cysteines in the CXXCH motif. Following addition of heme to reduced CXXCH apoprotein, spontaneous covalent addition of heme to polypeptide occurred in vitro. Strikingly, the spectral properties were very similar to those of the material obtained from cells in which presumed uncatalyzed addition of heme (i.e. in the absence of Ccm) had been observed. The major product from uncatalyzed heme attachment was an incorrectly matured cytochrome with the heme rotated by 180° relative to its normal orientation. The contrast between Ccm-dependent and Ccm-independent covalent attachment of heme indicates that the Ccm apparatus presents heme to the protein only in the orientation that results in formation of the correct product and also that heme does not become covalently attached to the apocytochrome b₅₆₂ CXXCH variant without being handled by the Ccm system in the periplasm. The CXXCH variant of cytochrome b₅₆₂ was also expressed in E. coli strains deficient in the periplasmic reductant DsbD or oxidant DsbA. In the DsbA^- strain under aerobic conditions, c-type cytochromes were made abundantly and correctly when the Ccm proteins were expressed. This contrasts with previous reports indicating that DsbA is essential for cytochrome c biogenesis in E. coli.

Received for publication, July 5, 2003 , and in revised form, September 30, 2003.

^* This work was supported in part by Grant C13443 (to S. J. F.) and an advanced fellowship (to P. D. B.) from the Biotechnology and Biological Sciences Research Council. 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1-S5.

A William R. Miller Junior Research Fellow (St. Edmund Hall, Oxford, United Kingdom).

^|| To whom correspondence should be addressed. Tel.: 44-1865-275-240; Fax: 44-1865-275-259; E-mail: stuart.ferguson{at}bioch.ox.ac.uk.

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