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Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the reversible exchange of N? for H?.

ABSTRACT: Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cooling the sample to cryogenic temperature for EPR analysis. During -50 °C cryoannealing of EG prepared under turnover conditions in which the concentrations of N2 and H2 ([H2], [N2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H2] and slowed by increasing [N2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H2] and/or high [N2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination of H2 with concomitant N2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N2 and two [e(-)/H(+)] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N2 and H2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3.

SUBMITTER: Lukoyanov D

PROVIDER: S-EPMC4374740 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the reversible exchange of N₂ for H₂.

Lukoyanov Dmitriy D Yang Zhi-Yong ZY Khadka Nimesh N Dean Dennis R DR Seefeldt Lance C LC Hoffman Brian M BM

Journal of the American Chemical Society 20150305 10

Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cool ...[more]

PMID: 25741750

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Project description:Nitrogenase reduces dinitrogen (N2) by six electrons and six protons at an active-site metallocluster called FeMo cofactor, to yield two ammonia molecules. Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N2, by wild-type MoFe protein, providing evidence that it contains N2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady-state turnover with N2, the S = 3/2 EPR signal (g = [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = 1/2, signal (g = [2.08, 1.99, 1.97]). The intensity of the N2-dependent EPR signal increased with increasing N2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at > or = 0.2 atm N2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein:FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. 15N ENDOR spectroscopic analysis of MoFe protein captured during turnover with 15N2 revealed a 15N nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A = [0.9, 1.4, 0.45] MHz establishing that an N2-derived species was trapped on the FeMo cofactor. The observation of a single type of 15N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable 1H ENDOR signal, is consistent with an N2 molecule bound end-on to the FeMo cofactor.

Dataset Information

Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the reversible exchange of N? for H?.

Publications

Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the reversible exchange of N₂ for H₂.

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