Nitrite and Hydroxylamine as Nitrogenase Substrates: Mechanistic Implications for the Pathway of N2 Reduction
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- 作者：Sudipta Shaw ; Dmitriy Lukoyanov ; Karamatullah Danyal ; Dennis R. Dean ; Brian M. Hoffman ; Lance C. Seefeldt
- 刊名：Journal of the American Chemical Society
- 出版年：2014
- 出版时间：September 10, 2014
- 年：2014
- 卷：136
- 期：36
- 页码：12776-12783
- 全文大小：527K
- ISSN：1520-5126
文摘
Investigations of reduction of nitrite (NO2鈥?/sup>) to ammonia (NH3) by nitrogenase indicate a limiting stoichiometry, NO2鈥?/sup> + 6e鈥?/sup> + 12ATP + 7H+ 鈫?NH3 + 2H2O + 12ADP + 12Pi. Two intermediates freeze-trapped during NO2鈥?/sup> turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N2 reduction. The proposed NO2鈥?/sup> reduction intermediate hydroxylamine (NH2OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped. Viewing N2 and NO2鈥?/sup> reductions in light of their common reduction intermediates and of NO2鈥?/sup> reduction by multiheme cytochrome c nitrite reductase (ccNIR) leads us to propose that NO2鈥?/sup> reduction by nitrogenase begins with the generation of NO2H bound to a state in which the active-site FeMo-co (M) has accumulated two [e鈥?/sup>/H+] (E2), stored as a (bridging) hydride and proton. Proton transfer to NO2H and H2O loss leaves M鈥揫NO+]; transfer of the E2 hydride to the [NO+] directly to form HNO bound to FeMo-co is one of two alternative means for avoiding formation of a terminal M鈥揫NO] thermodynamic 鈥渟ink鈥? The N2 and NO2鈥?/sup> reduction pathways converge upon reduction of NH2NH2 and NH2OH bound states to form state H with [鈭扤H2] bound to M. Final reduction converts H to I, with NH3 bound to M. The results presented here, combined with the parallels with ccNIR, support a N2 fixation mechanism in which liberation of the first NH3 occurs upon delivery of five [e鈥?/sup>/H+] to N2, but a total of seven [e鈥?/sup>/H+] to FeMo-co when obligate H2 evolution is considered, and not earlier in the reduction process.