PMID: 9173873Apr 1, 1997Paper

Cysteine-200 of human inducible nitric oxide synthase is essential for dimerization of haem domains and for binding of haem, nitroarginine and tetrahydrobiopterin

The Biochemical Journal
R R CubberleyR W Old

Abstract

Nitric oxide synthase (EC 1.14.13.39) is a homodimer. Limited proteolysis has previously shown that it consists of two major domains. The C-terminal or reductase domain binds FMN, FAD and NADPH. The N-terminal or oxygenase domain is known to bind arginine, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and haem. The exact residues of the inducible nitric oxide synthase (iNOS) protein involved in binding to these molecules have yet to be identified, although the haem moiety is known to be co-ordinated through a cysteine thiolate ligand. We have expressed two forms of the haem-binding domain of human iNOS (residues 1-504 and 59-504) in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The iNOS 1-504 and 59-504 fusion proteins bound similar amounts of haem, Nomega-nitro-l-arginine (nitroarginine) and tetrahydrobiopterin, showing that the first 58 residues are not required for binding these factors. Using site-directed mutagenesis we have mutated Cys-200, Cys-217, Cys-228, Cys-290, Cys-384 and Cys-457 to alanine residues within the iNOS 59-504 haem-binding domain. Mutation of Cys-200 resulted in a complete loss of haem, nitroarginine and tetrahydrobiopterin binding. Mutants of Cys-217, Cys-228, Cys-290...Continue Reading

Citations

Dec 4, 2013·Antioxidants & Redox Signaling·Jennifer K BendallMark J Crabtree
Feb 24, 2001·Antioxidants & Redox Signaling·M A TirmensteinM W Fariss
Nov 3, 2011·Chembiochem : a European Journal of Chemical Biology·Toni KühlDiana Imhof
Aug 22, 2000·The Journal of Biological Chemistry·A T BenderY Osawa
May 13, 1999·Biochimica Et Biophysica Acta·D J Stuehr
Jan 3, 2003·Protein Expression and Purification·Jennifer K McDonaldSteven Rafferty
Mar 20, 2003·Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP·Supatra PorasuphatanaGerald M Rosen

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