Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme

详细信息    查看全文

• 作者：Dmitriy Lukoyanov ; Nimesh Khadka ; Zhi-Yong Yang ; Dennis R. Dean ; Lance C. Seefeldt ; Brian M. Hoffman
• 刊名：Journal of the American Chemical Society
• 出版年：2016
• 出版时间：August 24, 2016
• 年：2016
• 卷：138
• 期：33
• 页码：10674-10683
• 全文大小：583K
• 年卷期：0
• ISSN：1520-5126

文摘

We proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N<sub>2sub> to 2NH<sub>3sub> by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70<sup>Val→Ilesup> MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe–H–Fe] bridging hydrides (denoted E<sub>4sub>(4H)). The mechanism postulates that obligatory re of the hydrides as H<sub>2sub> drives reduction of N<sub>2sub> to a state (denoted E<sub>4sub>(2N2H)) with a moiety at the diazene (HN═NH) reduction level bound to the catalytic FeMo-co. EPR/ENDOR/photophysical measurements on wild type (WT) MoFe protein now establish this mechanism. They show that a state freeze-trapped during N<sub>2sub> reduction by WT MoFe is the same Janus intermediate, thereby establishing the α-70<sup>Val→Ilesup> intermediate as a reliable guide to mechanism. Monitoring the Janus state in WT MoFe during N<sub>2sub> reduction under mixed-isotope condition, H<sub>2sub>O buffer/D<sub>2sub>, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby solving longstanding puzzles. Relaxation of E<sub>4sub>(2N2H) to the WT resting-state is shown to occur via oa of H<sub>2sub> and release of N<sub>2sub> to form Janus, followed by sequential release of two H<sub>2sub>, demonstrating the kinetic reversibility of the re/oa equilibrium. Relative populations of E<sub>4sub>(2N2H)/E<sub>4sub>(4H) freeze-trapped during WT turnover furthermore show that the reversible re/oa equilibrium between [E<sub>4sub>(4H) + N<sub>2sub>] and [E<sub>4sub>(2N2H) + H<sub>2sub>] is ∼ thermoneutral (Δ<sub>resub>G<sup>0sup> ∼ −2 kcal/mol), whereas, by itself, hydrogenation of N<sub>2sub>(g) is highly endergonic. These findings demonstrate that (i) re/oa accounts for the historical Key Constraints on mechanism, (ii) that Janus is central to N<sub>2sub> reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus, emerges a picture of the central mechanistic steps by which nitrogenase carries out one of the most challenging chemical transformations in biology.