Carbonyl sulfide and carbon dioxide as new substrates, and carbon disulfide as a new inhibitor, of nitrogenase.
Nitrogenase is the metalloenzyme responsible for the biological reduction of N2 to NH3. Nitrogenase has been shown to reduce a variety of substrates in addition to N2 and protons. General properties of alternative substrates for nitrogenase are the presence of N-N, N-O, N-C, and C-C triple or double bonds. In the present work, we demonstrate that Azotobacter vinelandii nitrogenase can reduce both C-S and C-O bonds. Nitrogenase was found to reduce carbonyl sulfide (COS), to CO and H2S at a maximum rate of 37.2 +/- 2.0 nmol min-1 (mg of protein)-1 with a Km of 3.1 +/- 0.6 mM. The formation of CO from nitrogenase reduction of COS was monitored spectrophotometrically in real time by following the formation of carboxyhemoglobin. In this assay, the change in the visible absorption spectrum of reduced hemoglobin upon binding CO provided a sensitive way to quantify CO formation and to remove CO, which is a potent inhibitor of nitrogenase, from solution. COS reduction by nitrogenase required the molybdenum-iron protein (MoFeP), the iron protein (FeP), and MgATP. The reduction reaction was inhibited by MgADP, acetylene, and N2, while H2 was not an inhibitor of COS reduction. The pH optimum for COS reduction was 6.5. Nitrogenase was also found to reduce carbon dioxide (CO2) to CO and H2O. CO2 was reduced at a maximum rate of 0.8 +/- 0.07 nmole min-1 (mg of protein)-1 with a calculated Km for CO2 of 23.3 +/- 3.7 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Seefeldt
L C
LC
Department of Chemistry and Biochemistry, Utah State University, Logan 84322-0300, USA.
Rasche
M E
ME
Ensign
S A
SA
D016948:Azotobacter vinelandii / Q000201:enzymology*; D002245:Carbon Dioxide / Q000378:metabolism*; D002246:Carbon Disulfide / Q000494:pharmacology*; D007700:Kinetics; D008962:Models, Theoretical; D009591:Nitrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity; D013461:Sulfur Oxides / Q000378:metabolism*
D013461:Sulfur Oxides; D002245:Carbon Dioxide; C010063:carbonyl sulfide; D009591:Nitrogenase; D002246:Carbon Disulfide