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J. Biol. Chem., Vol. 280, Issue 6, 4713-4721, February 11, 2005

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Identification and Characterization of the Terminal Enzyme of Siroheme Biosynthesis from Arabidopsis thaliana

A PLASTID-LOCATED SIROHYDROCHLORIN FERROCHELATASE CONTAINING A 2FE-2S CENTER^*

Evelyne Raux-Deery, Helen K. Leech, Kerry-Ann Nakrieko¶, Kirsty J. McLean||, Andrew W. Munro||**, Peter Heathcote, Stephen E. J. Rigby, Alison G. Smith, and Martin J. Warren

From the School of Biological Sciences, Queen Mary, University of London, Mile End Rd., London E1 4NS, Department of Plant Sciences, University of Cambridge, Downing St., Cambridge CB2 3EA, and ^||Department of Biochemistry, University of Leicester, University Rd., Leicester, LE1 7RH, United Kingdom

Higher plant sulfite and nitrite reductases contain siroheme as a prosthetic group. Siroheme is synthesized from the tetrapyrrole primogenitor uroporphyrinogen III in three steps involving methylation, oxidation, and ferrochelation reactions. In this paper we report on the Arabidopsis thaliana sirohydrochlorin ferrochelatase At-SirB. The complete precursor protein of 225 amino acids and shorter constructs in which the first 46 or 79 residues had been removed were shown to complement a defined Escherichia coli sirohydrochlorin ferrochelatase mutant. The mature form of the protein appeared to consist of only 150 amino acids, making it much smaller than previously characterized ferrochelatases. Green fluorescent protein tagging revealed that it is located in the chloroplast. The enzyme was easily produced in E. coli as a recombinant protein, and the isolated enzyme was found to have a specific activity of 48.5 nmol/min/mg. Significantly, the protein purified as a brown-colored solution with a UV-visible spectrum containing maxima at 415 and 455 nm, suggestive of an Fe-S center. EPR analysis of the recombinant protein produced a rhombic spectrum with G-values of 2.04, 1.94, and 1.90 and with temperature dependence consistent with a 2Fe-2S center. Redox titration demonstrated that the Fe-S center is highly unstable, with an apparent midpoint reduction potential of about -370 mV. This is the first Fe-S center to be reported in a higher plant ferrochelatase. The implications of the Fe-S center in an enzyme that is so closely associated with the metabolism of sulfur and iron are discussed.

Received for publication, October 5, 2004 , and in revised form, November 15, 2004.

^* This work was supported by grants from by the Biotechnology and Biological Sciences Research Council, the Wellcome Trust, and the European Community. 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.

^¶ Receipt of a studentship from the Cambridge Commonwealth Trust.

^** Recipient of a Royal Society Leverhulme Trust Senior Research Fellowship.

To whom correspondence should be addressed. Tel.: 20-7882-7718; Fax: 20-7882-7609; E-mail: m.j.warren{at}qmul.ac.uk.

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