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 N-2 to NH3. Nitrogenase has been shown to reduce a variety of substrates in addition to N-2 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 K-m 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 N-2, while H-2 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 K-m for CO2 of 23.3 +/- 3.7 mM. COS and CO2 did not inhibit proton reduction by nitrogenase or total electron flow through nitrogenase. In contrast, the COS and CO2 analog carbon disulfide (CS2) was found to be an inhibitor of nitrogenase reduction reactions and total electron flow. CS2 was a rapid-equilibrium, mixed-type inhibitor with respect to acetylene reduction (K-i1 = 2.9 +/- 0.6 mM and K-i2 = 12.1 +/- 2 mM) and also inhibited both proton and COS reduction.