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Structures of the N^ω-Hydroxy-l-Arginine Complex of Inducible Nitric Oxide Synthase Oxygenase Dimer with Active and Inactive Pterins

Crane, Brian R. and Arvai, Andrew S. and Ghosh, Sanjay and Getzoff, Elizabeth D. and Stuehr, Dennis J. and Tainer, John A. (2000) Structures of the N^ω-Hydroxy-l-Arginine Complex of Inducible Nitric Oxide Synthase Oxygenase Dimer with Active and Inactive Pterins. Biochemistry, 39 (16). pp. 4608-4621. ISSN 0006-2960. doi:10.1021/bi992409a.

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Nitric oxide synthases (NOSs) catalyze two mechanistically distinct, tetrahydrobiopterin (H4B)-dependent, heme-based oxidations that first convert l-arginine (l-Arg) to N^ω-hydroxy-l-arginine (NHA) and then NHA to l-citrulline and nitric oxide. Structures of the murine inducible NOS oxygenase domain (iNOS_(ox)) complexed with NHA indicate that NHA and l-Arg both bind with the same conformation adjacent to the heme iron and neither interacts directly with it nor with H_4B. Steric restriction of dioxygen binding to the heme in the NHA complex suggests either small conformational adjustments in the ternary complex or a concerted reaction of dioxygen with NHA and the heme iron. Interactions of the NHA hydroxyl with active center β-structure and the heme ring polarize and distort the hydroxyguanidinium to increase substrate reactivity. Steric constraints in the active center rule against superoxo-iron accepting a hydrogen atom from the NHA hydroxyl in their initial reaction, but support an Fe(III)-peroxo-NHA radical conjugate as an intermediate. However, our structures do not exclude an oxo-iron intermediate participating in either l-Arg or NHA oxidation. Identical binding modes for active H_4B, the inactive quinonoid-dihydrobiopterin (q-H_2B), and inactive 4-amino-H_4B indicate that conformational differences cannot explain pterin inactivity. Different redox and/or protonation states of q-H_2B and 4-amino-H_4B relative to H_4B likely affect their ability to electronically influence the heme and/or undergo redox reactions during NOS catalysis. On the basis of these structures, we propose a testable mechanism where neutral H_4B transfers both an electron and a 3,4-amide proton to the heme during the first step of NO synthesis.

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Additional Information:© 2000 American Chemical Society. Received 18 October 1999. Published online 31 March 2000. Published in print 1 April 2000. Supported by National Institute of Health Grants HL58883 (E.D.G.) and CA53914 (D.J.S.), fellowship grants from the Helen Hay Whitney Foundation (B.R.C.), and the Skaggs Institute for Research (A.S.A. and B.R.C.). We thank the Advanced Light Source (ALS) and the Stanford Synchrotron Radiation Laboratory (SSRL) for use of data collection facilities, B. Mayer for 4-amino-H4B, R. J. Rosenfeld for assistance with data collection, and A.M. Bilwes for helpful discussions.
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Helen Hay Whitney FoundationUNSPECIFIED
Skaggs Institute for ResearchUNSPECIFIED
Issue or Number:16
Record Number:CaltechAUTHORS:20170201-091759564
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Official Citation:Structures of the Nω-Hydroxy-l-Arginine Complex of Inducible Nitric Oxide Synthase Oxygenase Dimer with Active and Inactive Pterins Brian R. Crane, Andrew S. Arvai, Sanjay Ghosh, Elizabeth D. Getzoff, Dennis J. Stuehr, and John A. Tainer Biochemistry 2000 39 (16), 4608-4621 DOI: 10.1021/bi992409a
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73915
Deposited By: Ruth Sustaita
Deposited On:01 Feb 2017 18:07
Last Modified:11 Nov 2021 05:23

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