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 13C 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, 13CS2 was used as a substrate, and freeze. trapped turnover samples were examined by Q-band 13C ENDOR spectroscopy. Each EPR signal ('a', 'b', and 'c') gave rise to a distinct 13C signal, with hyperfine coupling constants of 4.9 MHz for 13C(a), 1.8 MHz for 13C(b), and 2.7 MHz for 13C(c). Models for the sequential formation of intermediates during nitrogenase reduction of CS2 are discussed.
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