Distal Val346Ile Mutation in Inducible NO Synthase Promotes Substrate-Dependent NO Confinement

详细信息    查看全文

• 作者：Edward Beaumont ; Jean-Christophe Lambry ; Zhi-Qiang Wang ; Dennis J. Stuehr ; Jean-Louis Martin ; Anny Slama-Schwok
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：November 27, 2007
• 年：2007
• 卷：46
• 期：47
• 页码：13533 - 13540
• 全文大小：216K
• 年卷期：v.46,no.47(November 27, 2007)
• ISSN：1520-4995

文摘

The function of inducible NO synthase (WT iNOS) depends on the release of NO from theferric heme before the enzyme is reduced. Key parameters controlling ligand dynamics include the distaland proximal heme pocket amino acids, as well as the inner solvent molecules. In this work, we testedhow a point mutation in the distal heme side of WT iNOS affected the geminate rebinding of NO byultrafast kinetics and molecular dynamics simulations. The mutation sequestered much of the photodissociated NO close to the heme compared to WT iNOS, with a main picosecond phase accounting for78% of the rebinding to the arginine-bound Val346Ile protein. Consequently, the probability of NO releasefrom Val346Ile decreased as compared to that from WT iNOS, provided the substrate binding site isfilled. These data are rationalized by a steric effect of the Ile methyl group inducing events mediated bythe substrate, transmitted via the propionates to the NO and the protein. This model is consistent with therole of the H-bonding network involving the heme, the substrate, and the BH₄ cofactor in controlling NOrelease, with a key role of the heme propionates [Gautier et al. (2006) Nitric Oxide 15, 312]. These datasupport the effect of Val346Ile mutation in decreasing NO release and slowing down NO synthesiscompared to WT iNOS determined by single turnover catalysis [Wang et al. (2004) J. Biol. Chem. 279,19018].