Identification and characterization of the terminal enzyme of siroheme biosynthesis from Arabidopsis thaliana: a plastid-located sirohydrochlorin ferrochelatase containing a 2FE-2S center

• Published in: The Journal of biological chemistry 2005-02, Vol.280 (6), p.4713-4721
• Main Authors: Raux-Deery, Evelyne, Leech, Helen K, Nakrieko, Kerry-Ann, McLean, Kirsty J, Munro, Andrew W, Heathcote, Peter, Rigby, Stephen E J, Smith, Alison G, Warren, Martin J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - chemistry; Arabidopsis - metabolism; Chloroplasts - metabolism; DNA, Complementary - metabolism; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Escherichia coli - metabolism; Ferrochelatase - chemistry; Gene Library; Genetic Complementation Test; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - metabolism; Heme - analogs & derivatives; Heme - biosynthesis; Heme - chemistry; Iron-Sulfur Proteins - chemistry; Kinetics; Models, Biological; Models, Chemical; Molecular Sequence Data; Oxidation-Reduction; Plant Proteins - chemistry; Plasmids - metabolism; Plastids - metabolism; Potentiometry; Protein Binding; Sequence Homology, Amino Acid; Temperature; Ultraviolet Rays; Uroporphyrins - chemistry
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M411360200

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.