The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg2+ chelatase, such as the ATPase activity of ChlI, and the formation of ChlI–ChlD–MgATP and ChlH–protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg2+ chelatase activity, and the formation of the ChlI–ChlD–ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI–ChlD–MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg2+ ions. The simplest explanation for this is that in an intact Mg2+ chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI–MgATP association, ATP hydrolysis and in the interaction of ChlH with MgATP and protoporphyrin IX.

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