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| The EMBO Journal
(1999) 18, 6260–6270, doi:10.1093/emboj/18.22.6260 |
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Inducible nitric oxide synthase: role of the N-terminal  -hairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction |
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| Dipak K. Ghosh1, 2, Brian R. Crane3, 4, Sanjay Ghosh1, Dennis Wolan1, Ratan Gachhui1, Carol Crooks1, Anthony Presta1, John A. Tainer3, Elizabeth D. Getzoff3 and Dennis J. Stuehr1 |
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1 Department of Immunology, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH 44195, USA 2 Present address: Department of Medicine, Duke University and VA Medical Center, Durham, NC 27705, USA 3 Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 4 Present address: Department of Chemistry, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA To whom correspondence should be addressed Dipak K. Ghosh, stuehrd@ccf.org Dennis J. Stuehr, dgx@acpub.duke.edu Received 18 August 1999; Revised 30 September 1999; Accepted 30 September 1999. |
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| Abstract |
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| The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1–498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H4B) and is the site for nitric oxide synthesis. We examined an N-terminal segment that contains a -hairpin hook, a zinc ligation center and part of the H4B-binding site for its role in dimerization, catalysis, and H4B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N-terminal -hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side-chain hydrogen bonds and packing interactions with the iNOSox C-terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H4B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C-terminus and cause similar defects that were reversible with high concentrations of H4B. Heterodimers comprised of a hook-defective iNOSox mutant subunit and a full-length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N-terminal hooks exchange between subunits in solution to stabilize the dimer. |
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| Keywords: dimer, -hairpin hook, heme protein, nitric oxide, oxidoreductase |
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