Báo cáo khoa học: Comparing the substrate specificities of cytochrome c biogenesis Systems I and II

Comparing the substrate specificities of cytochrome c

biogenesis Systems I and II

Bioenergetics

Alan D. Goddard*, Julie M. Stevens*, Arnaud Rondelet, Elena Nomerotskaia, James W. A. Allen

and Stuart J. Ferguson

Department of Biochemistry, University of Oxford, UK

Introduction

Nature employs at least five distinct systems for the

biogenesis of c-type cytochromes [1–3]; this post-trans￾lational modification process covalently links the heme

cofactor to, normally, two cysteines in a CXXCH

motif. System I is found in many Gram-negative bacte￾ria and various mitochondria, including from plants

[4,5]; System II appears in Gram-positive and some

Gram-negative bacteria, and chloroplasts [6]; System

III occurs in many non-plant mitochondria [5]; System

IV is specific for the unusual cytochrome b6 involved

in photosynthesis [7], and a fifth system, which remains

to be characterized, exists in trypanosomatids [8]. Very

unusually, some thermophilic cytochromes c are able

to form spontaneously in vitro or in the cytoplasm of

Escherichia coli [9], although it is believed that they are

naturally matured by one of the biogenesis systems

above.

The experimental amenability of E. coli has allowed

the heterologous replacement of its own cytochrome

c maturation (Ccm) machinery (encoded by the

ccmABCDEFGH operon, called System I, Fig. 1) with

systems from other organisms to facilitate their analy￾sis. The enzyme heme lyase (System III) has been

shown to function in E. coli cytoplasm [10] and to

Keywords

cytochrome c; cytochrome c maturation;

heme; heme provision; System II

Correspondence

S. J. Ferguson, Department of

Biochemistry, University of Oxford, South

Parks Road, Oxford OX1 3QU, UK

Fax: +44 1865 613201

Tel: +44 1865 613299

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 28 July 2009, revised 12 October

2009, accepted 25 November 2009)

doi:10.1111/j.1742-4658.2009.07517.x

c-Type cytochromes require specific post-translational protein systems,

which vary in different organisms, for the characteristic covalent attach￾ment of heme to the cytochrome polypeptide. Cytochrome c biogenesis

System II, found in chloroplasts and many bacteria, comprises four subun￾its, two of which (ResB and ResC) are the minimal functional unit. The

ycf5 gene from Helicobacter pylori encodes a fusion of ResB and ResC.

Heterologous expression of ResBC in Escherichia coli lacking its own bio￾genesis machinery allowed us to investigate the substrate specificity of Sys￾tem II. ResBC is able to attach heme to monoheme c-type cytochromes

c550 from Paracoccus denitrificans and c552 from Hydrogenobacter thermo￾philus, both normally matured by System I. The production of holocyto￾chrome is enhanced by the addition of exogenous reductant. Single-cysteine

variants of these cytochromes were not efficiently matured by System II,

but System I was able to produce detectable amounts of AXXCH variants;

this adds to evidence that there is no obligate requirement for a disulfide￾bonded intermediate for the latter c-type cytochrome biogenesis system. In

addition, System II was able to mature an AXXAH-containing variant into

a b-type cytochrome, with implications for both heme supply to the peri￾plasm and substrate recognition by System II.

Abbreviations

Ccm, cytochrome c maturation; IPTG, isopropyl thio-b-D-galactoside; MESA, 2-mercaptoethane sulfonate.

726 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS