Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii

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Abstract
The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of S. itersonii .apprx. 490-fold. Purification was achieved by solubilization with chaotropic salts followed by (NH4)2 SO4 fractionation, DEAE cellulose chromatography and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum MW of .apprx. 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-MW aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of Cu. The apparent Km for Fe2+ is 20 .mu.M in the absence of Cu and 9.5 .mu.M in the presence of 20 .mu.M CuCl2. The apparent Km for protoporphyrin is 50 .mu.M, and it is unaltered by Cu. Ferrochelatase has a single pH optimum of 7.50, and it is inhitibed 50% by 20 .mu.M heme. Certain divalent cations and SH reagents also inhibit the enzyme.

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