Nitrogenase reduction of carbon disulfide: Freeze-quench EPR and ENDOR evidence for three sequential intermediates with cluster-bound carbon moieties

Freeze-quenching of nitrogenase during reduction of carbon disulfide (CS2) was previously shown to result in the appearance of a novel EPR signal (g = 2.21, 1.99, and 1.97) not previously associated with any of the oxidation states of the nitrogenase metal clusters. In the present work, freeze-quench X-and Q-band EPR and Q-band C-13 electron nuclear double resonance (ENDOR) spectroscopic studies of nitrogenase during CS2 reduction disclose the sequential formation of three distinct intermediates with a carbon-containing fragment of CS2 bound to a metal cluster inferred to be the molybdenum-iron cofactor. Modeling of the Q-band (35 GHz) EPR spectrum of freeze-trapped samples of nitrogenase during turnover with CS2 allowed assignment of three signals designated a (g = 2.035, 1.982, 1.973), b (g = 2.111, 2.002, and 1.956), and c (g = 2,211, 1.996, and 1.978). Freezing samples at varying times after initiation of the reaction reveals that signals a, b, and c appear and disappear in sequential order. Signal a reaches a maximal intensity at 25 s; signal b achieves maximal intensity at 60 s; and signal ''c shows maximal intensity at 100 s, To characterize the intermediates, (CS2)-C-13 was used as a substrate, and freeze-trapped turnover samples were examined by Q-band C-13 ENDOR spectroscopy. Each EPR signal (a, b, and c) gave rise to a distinct C-13 signal, with hyperfine coupling constants of 4.9 MHz for C-13(a), 1.8 MHz for C-13(b), and 2.7 MHz for C-13(c). Models for the sequential formation of intermediates during nitrogenase reduction of CS2 are discussed.