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 (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.
Ryle
M J
MJ
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
Lee
H I
HI
Seefeldt
L C
LC
Hoffman
B M
BM
D016948:Azotobacter vinelandii / Q000201:enzymology; D002244:Carbon / Q000737:chemistry*; D002246:Carbon Disulfide / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005615:Freezing; D008983:Molybdoferredoxin / Q000737:chemistry; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D013379:Substrate Specificity
D002247:Carbon Isotopes; D008983:Molybdoferredoxin; D002244:Carbon; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D002246:Carbon Disulfide