A Confirmationof the Quench-Cryoannealing RelaxationProtocol for Identifying Reduction States of Freeze-Trapped NitrogenaseIntermediates

摘要

We have advanced a mechanism for nitrogenase catalysis that rests on the identification of a low-spin EPR signal (S = 1/2) trapped during turnover of a MoFe protein as the E4 state, which has accumulated four reducing equivalents as two [Fe–H–Fe] bridging hydrides. Because electrons are delivered to the MoFe protein one at a time, with the rate-limiting step being the off-rate of oxidized Fe protein, it is difficult to directly control, or know, the degree of reduction, n, of a trapped intermediate, denoted En, n = 1–8. To overcome this previously intractable problem, we introduced a quench-cryoannealing relaxation protocol for determining n of an EPR-active trapped En turnover state. The trapped “hydride” state was allowed to relax to the resting E0 state in frozen medium, which prevents additional accumulation of reducing equivalents; binding of reduced Fe protein and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. Relaxation of En was monitored by periodic EPR analysisat cryogenic temperature. The protocol rests on the hypothesis thatan intermediate trapped in the frozen solid can relax toward the restingstate only by the release of a stable reduction product from FeMo-co.In turnover under Ar, the only product that can be released is H2, which carries two reducing equivalents. This hypothesisimplicitly predicts that states that have accumulated an odd numberof electrons/protons (n = 1, 3) during turnover underAr cannot relax to E0: E3 can relax to E1, but E1 cannot relax to E0 in the frozenstate. The present experiments confirm this prediction and, thus,the quench-cryoannealing protocol and our assignment of E4, the foundation of the proposed mechanism for nitrogenase catalysis.This study further gives insights into the identity of the En intermediates with high-spin EPR signals, 1b and1c, trapped under high electron flux.