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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December 2000
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Research Article|
November 24 2000
Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation
Poul E. JENSEN;
Poul E. JENSEN
*Department of Plant Biology, Plant Biochemistry Laboratory, The Royal Veterinary and Agricultural University, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark
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James D. REID;
James D. REID
*Department of Plant Biology, Plant Biochemistry Laboratory, The Royal Veterinary and Agricultural University, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark
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C. Neil HUNTER
C. Neil HUNTER
1
†Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
1To whom correspondence should be addressed (e-mail c.n.hunter@sheffield.ac.uk).
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Publisher: Portland Press Ltd
Received:
May 22 2000
Revision Received:
June 21 2000
Accepted:
September 14 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 352 (2): 435–441.
Article history
Received:
May 22 2000
Revision Received:
June 21 2000
Accepted:
September 14 2000
Citation
Poul E. JENSEN, James D. REID, C. Neil HUNTER; Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation. Biochem J 1 December 2000; 352 (2): 435–441. doi: https://doi.org/10.1042/bj3520435
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