A Key Piece in the Global N-Cycle: The N–N Bond Formation Presented by Heme-Dependent Hydrazine Synthase
Jing‐Xuan Su, Shi‐Lu Chen
Abstract
Responsible for the critical step of N–N bond formation in the global N-cycle, hydrazine synthase (HZS), a heme-dependent enzyme, catalyzes the production of hydrazine (N2H4) from NO and NH3. Herein, the HZS reaction is demonstrated to be divided into two half reactions, that is, the reduction of NO to NH2OH with protons and electrons added and the condensation of NH2OH with NH3 forming N2H4. The first half reaction is assessed to be thermodynamically feasible, and the second half reaction is revealed, by density functional calculations based on a crystal-structure-derived chemical model, to proceed via a redox mechanism mainly in the singlet state, including the homolytic N–O bond cleavage of NH2OH activated by FeII-heme αI yielding FeIII–NH2 and •OH radicals, the hydrogen-atom transfer from NH3 to •OH forming a •NH2 radical and a water, and the N–N bond formation between two •NH2 radicals. The hydrogen transfer is rate-limiting with an overall barrier of 22.1 kcal/mol. Other competing mechanisms starting from different substrate-binding modes or a FeIII state have been ruled out. NH4+ is most unlikely to directly participate in the HZS reaction with unfavorable energetics and must be stripped of a proton by the Zn site near to heme αI and/or the hydrophobic active site, ensuring the participation of a neutral NH3. A protonated FeII-heme could not catalyze the N2H4 synthesis. The work here gives deep insights into the fundamental chemistry of enzymatic N–N bond formation and also enriches the understanding of heme chemistry.