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Ryle MJ, Lee HI, Seefeldt LC, Hoffman BM: Nitrogenase reduction of carbon disulfide: freeze-quench EPR and ENDOR evidence for three sequential intermediates with cluster-bound carbon moieties. Biochemistry. 2000 Feb 8;39(5):1114-9.
Freeze-quenching of nitrogenase during reduction of carbon disulfide (CS (2)) 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 (13) C electron nuclear double resonance (ENDOR) spectroscopic studies of nitrogenase during CS (2) reduction disclose the sequential formation of three distinct intermediates with a carbon-containing fragment of CS (2) 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 CS (2) 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, (13) CS (2) was used as a substrate, and freeze-trapped turnover samples were examined by Q-band (13) C ENDOR spectroscopy. Each EPR signal ("a", "b", and "c") gave rise to a distinct (13) C signal, with hyperfine coupling constants of 4.9 MHz for (13) C (a), 1. 8 MHz for (13) C (b), and 2.7 MHz for (13) C (c). Models for the sequential formation of intermediates during nitrogenase reduction of CS (2) are discussed. |
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