Abstract
Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k(cat) and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) and can be alleviated by the addition of metal complexing agents such as beta-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.
Highlights
Ferrochelatase (EC 4.99.1.1) catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme [1, 2]
At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts ferrous iron and divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability
Mutations in the human ferrochelatase gene have been linked to erythropoietic protoporphyria, a disease characterized by protoporphyrin IX accumulation and acute photosensitivity [3]
Summary
Reagents—MOPS, Tween 80, sodium chloride, cobalt chloride hexahydrate, zinc chloride, nickel chloride hexahydrate, and cupric chloride dihydrate were from Sigma. Preparation of Protoporphyrin IX and Metal Ion Solutions— Stock solutions of protoporphyrin IX were prepared from the commercially available hydrochloride salt by vortexing 1–3 mg in 100 l of 2 M sodium hydroxide This was followed by the sequential addition of 0.5 ml of 10% (w/v) Tween 80, 4 ml of deionized water, 90 l of 2 M hydrochloric acid, and 0.5 ml of 1 M MOPS, pH 7.00, with thorough mixing after each addition. Estimation of Extinction Coefficients—Extinction coefficients for protoporphyrin IX in ferrochelatase reaction buffer at 30 °C were determined by observation of the signal amplitudes produced from a stock solution prepared as described above ( see supplemental Fig. 2). Apparent extinction coefficients for conversion of protoporphyrin IX to metallated protoporphyrins in this buffer were determined in a similar fashion using 0.2 M yeast ferrochelatase and excess metal ion to convert known concentrations of protoporphyrin IX into the corresponding metalloporphyrins. Spectral changes associated with the metallation reactions are provided as supplemental Fig. 3
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