Ferrochelatase from Rhodopseudomonas sphaeroides: substrate specificity and role of sulfhydryl and arginyl residues

Abstract

Purified ferrochelatase (protoheme ferrolyase; EC 4.99.1.1) from the bacterium Rhodopseudomonas sphaeroides was examined to determine the roles of cationic and sulfhydryl residues in substrate binding. Reaction of the enzyme sulfhydryl residues with N-ethylmaleimide or monobromobimane resulted in a rapid loss of enzyme activity. Ferrous iron, but not porphyrin substrate, had a protective effect against inactivation by these two reagents. Quantitation with 3H-labeled N-ethylmaleimide revealed that inactivation required one to two sulfhydryl groups to be modified. Modification of arginyl residues with either 2,3-butanedione or camphorquinone 10-sulfonate resulted in a loss of ferrochelatase activity. A kinetic analysis of the modified enzyme showed that the Km for ferrous iron was not altered but that the Km for the porphyrin substrate was increased. These data suggested that arginyl residues may be involved in porphyrin binding, possibly via charge pair interactions between the arginyl residue and the anionic porphyrin propionate side chain. Modification of lysyl residues had no effect on enzyme activity. We also examined the ability of bacterial ferrochelatase to use various 2,4-disubstituted porphyrins as substrates. We found that 2,4-bis-acetal-and 2,4-disulfonate deuteroporphyrins were effective substrates for the purified bacterial enzyme and that N-methylprotoporphyrin was an effective inhibitor of the enzyme. Our data for the ferrochelatase of R. sphaeroides are compared with previously published data for the eucaryotic enzyme.

Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts

“…The enzyme catalyzing the insertion of iron into Proto, ferrochelatase, has been well characterized in bacterial (11,19), mammalian (8)(9)(10), and to some extent in plant systems (20). The reaction is clearly very similar to that catalyzed by .…”

“…Surprisingly, the only porphyrins which were found to inhibit Mg-chelatase were NMPP and NMMP (Fig. 5) which, up to this point, have been considered to be specific ferrochelatase inhibitors (10,11). Although these compounds have been reported to have no effect on Chl synthesis (and presumably Mg-chelatase) in vivo (2,6,19) (10).…”

“…This lack of specificity at the 2-and 4-positions of the porphyrin is common in enzymes in this portion of the pathway. The methyl transferase, the Mg-Proto monomethyl ester cyclase (28), Pchlide reductase (CJ Walker, PA Castelfranco, personal observation) as well as ferrochelatase (10,11,20) have all been shown to share this flexible specificity to some extent. The implication of this work is that probably none of these enzymes absolutely require the 2-and 4-vinyl groups for substrate binding.…”

The biosynthesis of porphyrins, chlorophylls, and vitamin B12

“…At some stage in the biosynthesis, ring-contraction occurs to generate the corrin macrocycle, and accordingly the intermediates after this stage will be called 'corrins'. For example, if (96) proves to be an intermediate, it will be called corrin-6 (or possibly corrin-6A). This nomenclature, if used properly, should prevent any future confusion over structures and it will be adopted in future reviews in this series.…”