The Nitric Oxide Reductase Mechanism of a Flavo-Diiron Protein: Identification of Active-Site Intermediates and Products

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• 作者：Jonathan D. Caranto ; Andrew Weitz ; Michael P. Hendrich ; Donald M. Kurtz ; Jr.
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：June 4, 2014
• 年：2014
• 卷：136
• 期：22
• 页码：7981-7992
• 全文大小：624K
• 年卷期：v.136,no.22(June 4, 2014)
• ISSN：1520-5126

文摘

The unique active site of flavo-diiron proteins (FDPs) consists of a nonheme diiron-carboxylate site proximal to a flavin mononucleotide (FMN) cofactor. FDPs serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria, archaea, and some protozoan parasites. Nitric oxide is reduced to nitrous oxide by the four-electron reduced (FMNH₂鈥揊ep>IIp>Fep>IIp>) active site. In order to clarify the nitric oxide reductase mechanism, we undertook a multispectroscopic presteady-state investigation, including the first M枚ssbauer spectroscopic characterization of diiron redox intermediates in FDPs. A new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous-dinitrosyl (S = 0, [{FeNO}p>7p>]₂) species. This species has an exchange energy, J 鈮?40 cmp>鈥?p> (JS₁ 掳 S₂), which is consistent with a hydroxo or oxo bridge between the two irons. The results show that the nitric oxide reductase reaction proceeds through successive formation of diferrous-mononitrosyl (S = p>1p>/₂, Fep>IIp>{FeNO}p>7p>) and the S = 0 diferrous-dinitrosyl species. In the rate-determining process, the diferrous-dinitrosyl converts to diferric (Fep>IIIp>Fep>IIIp>) and by inference N₂O. The proximal FMNH₂ then rapidly rereduces the diferric site to diferrous (Fep>IIp>Fep>IIp>), which can undergo a second 2NO 鈫?N₂O turnover. This pathway is consistent with previous results on the same deflavinated and flavinated FDP, which detected N₂O as a product (p person-group-type="allauthors">Hayaship> Biochemistrypan class="NLM_x"> pan>2010pan class="NLM_x">, pan>49pan class="NLM_x">, pan>7040). Our results do not support other proposed mechanisms, which proceed either via 鈥渟uper-reduction鈥?of [{FeNO}p>7p>]₂ by FMNH₂ or through Fep>IIp>{FeNO}p>7p> directly to a diferric-hyponitrite intermediate. The results indicate that an S = 0 [{FeNO}p>7p>}]₂ complex is a proximal precursor to N鈥揘 bond formation and N鈥揙 bond cleavage to give N₂O and that this conversion can occur without redox participation of the FMN cofactor.