Howard Hughes Medical Institute, Department of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California91125, United States.
J Am Chem Soc. 2022 Nov 23;144(46):21125-21135. doi: 10.1021/jacs.2c07574. Epub 2022 Nov 8.
The mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, has been of great interest for understanding the catalytic strategy utilized to reduce dinitrogen to ammonia under ambient temperatures and pressures. The reduction mechanism of nitrogenase is generally envisioned as involving multiple cycles of electron and proton transfers, with the known substrates requiring at least two cycles. Solvent kinetic isotope effect experiments, in which changes of reaction rates or product distribution are measured upon enrichment of solvent with heavy atom isotopes, have been valuable for deciphering the mechanism of complex enzymatic reactions involving proton or hydrogen transfer. We report the distribution of ethylene, dihydrogen, and methane isotopologue products measured from nitrogenase-catalyzed reductions of acetylene, protons, and cyanide, respectively, performed in varying levels of deuterium enrichment of the solvent. As has been noted previously, the total rate of product formation by nitrogenase is largely insensitive to the presence of DO in the solvent. Nevertheless, the incorporation of H/D into products can be measured for these substrates that reflect solvent isotope effects on hydrogen atom transfers that are faster than the overall rate-determining step for nitrogenase. From these data, a minimal isotope effect is observed for acetylene reduction (1.4 ± 0.05), while the isotope effects for hydrogen and methane evolution are significantly higher at 4.2 ± 0.1 and 4.4 ± 0.1, respectively. These results indicate that there are pronounced differences in the sensitivity to isotopic substitution of the hydrogen atom transfer steps associated with the reduction of these substrates by nitrogenase.
固氮酶的作用机制一直是人们研究的热点，因为它能够在常温常压下将氮气还原为氨。固氮酶的还原机制通常涉及多个电子和质子转移循环，已知的底物至少需要两个循环。溶剂动力学同位素效应实验通过富集重原子同位素来测量反应速率或产物分布的变化，对于解析涉及质子或氢转移的复杂酶反应机制非常有价值。我们报告了在不同程度的溶剂氘富集条件下，固氮酶催化乙炔、质子和氰化物还原反应生成的乙烯、氢气和甲烷同位素产物的分布。如前所述，固氮酶的总产物形成速率对溶剂中 DO 的存在基本不敏感。然而，对于这些反映氢原子转移溶剂同位素效应快于固氮酶总速率决定步骤的底物，可以测量 H/D 掺入产物的情况。从这些数据中，乙炔还原的最小同位素效应为 1.4 ± 0.05，而氢气和甲烷生成的同位素效应分别显著更高，为 4.2 ± 0.1 和 4.4 ± 0.1。这些结果表明，固氮酶还原这些底物时，与氢原子转移步骤相关的同位素取代敏感性存在显著差异。
