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-quenc h X-and Q-band EPR and Q-band C-13 electron nuclear double resonance (ENDOR ) spectroscopic studies of nitrogenase during CS2 reduction disclose the se quential formation of three distinct intermediates with a carbon-containing fragment of CS2 bound to a metal cluster inferred to be the molybdenum-iro n 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, a nd 1.956), and "c" (g = 2,211, 1.996, and 1.978). Freezing samples at varyi ng times after initiation of the reaction reveals that signals "a", "b", an d "c" appear and disappear in sequential order. Signal "a" reaches a maxima l intensity at 25 s; signal "b" achieves maximal intensity at 60 s; and sig nal ''c" shows maximal intensity at 100 s, To characterize the intermediate s, (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 cons tants 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 re duction of CS2 are discussed.