CYSTEINE-200 OF HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE IS ESSENTIAL FOR DIMERIZATION OF HEME DOMAINS AND FOR BINDING OF HEME, NITROARGININE AND TETRAHYDROBIOPTERIN

Nitric oxide synthase (EC 1.14.13.39) is a homodimer. Limited proteoly sis 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 i s known to be co-ordinated through a cysteine thiolate ligand. We have expressed two forms of the haem-binding domain of human iNOS (residue s 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, N-omega-nitro-L-arginine (nitroarginine) and tetrahydrobiopterin, showing that the first 58 residues are not requir ed 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 ala nine residues within the iNOS 59-504 haem-binding domain. Mutation of Cys-200 resulted in a complete loss of haem, nitroarginine and tetrahy drobiopterin 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, n o effect on the reduced CO spectral peak (444 nm) and were able to bin d nitroarginine and tetrahydrobiopterin at levels equivalent to the wi ld-type fusion protein. After removal of the GST polypeptide, the wild -type iNOS 59-504 domain was dimeric, whereas the C200A mutant form wa s monomeric. When the mutated domains were incorporated into a reconst ructed full-length INOS protein expressed in Xenopus oocytes, only the Cys-200 mutant showed a loss of catalytic activity: all the other mut ant iNOS proteins showed near wild-type enzymic activity. From this sy stematic 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 activit y of iNOS, and that Cys-200 provides the proximal thiolate ligand for haem binding in human iNOS.