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, Nω-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, Cys-384 or Cys-457 showed no effect on the haem content of the fusion protein, no effect on the reduced CO spectral peak (444 nm) and were able to bind nitroarginine and tetrahydrobiopterin at levels equivalent to the wild-type fusion protein. After removal of the GST polypeptide, the wild-type iNOS 59-504 domain was dimeric, whereas the C200A mutant form was monomeric. When the mutated domains were incorporated into a reconstructed full-length iNOS protein expressed in Xenopus oocytes, only the Cys-200 mutant showed a loss of catalytic activity: all the other mutant iNOS proteins showed near wild-type enzymic activity. From this systematic approach we conclude that although Cys-217, Cys-228, Cys-290, Cys-384 and Cys-457 are conserved in all three NOS isoforms they are not essential for cofactor or substrate binding or for enzymic activity of iNOS, and that Cys-200 provides the proximal thiolate ligand for haem binding in human iNOS.

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