Reduction of thiocyanate, cyanate, and carbon disulfide by nitrogenase: kinetic characterization and EPR spectroscopic analysis.

Nitrogenase catalyzes the reduction of N2, protons, and a number of alternative substrates that contain C-C, C-N, N-N, and N-O double and triple bonds. Recently it has been shown that nitrogenase also reduces the C==S bond of COS and the C==O bond of CO2. The current work demonstrates that the COS analogs SCN-, CS2, and OCNH are novel substrates for nitrogenase and that the reduction of these substrates produces changes in the electron paramagnetic resonance (EPR) spectrum of nitrogenase, providing insight into the mechanism of substrate reduction by nitrogenase. CH4, HCN, H2S, and NH4+ were detected as products of the nitrogenase-catalyzed reduction of SCN-. CS2 was reduced by nitrogenase to H2S, providing the first demonstration of CS2 reduction catalyzed by a purified enzyme. CO was detected as a product of KOCN reduction by nitrogenase. Interestingly, the Km for KOCN reduction to CO decreased at lower pH values, suggesting that OCNH rather than OCN- was the substrate for nitrogenase. Analysis of the EPR spectra of nitrogenase under turnover conditions in the presence of KOCN, CS2, or KSCN revealed new EPR signals. Signals with g-values corresponding to those reported for CO bound to the iron-molybdenum cofactor of nitrogenase were detected during turnover of nitrogenase in the presence of KOCN. During SCN- and CS2 reduction by nitrogenase, novel EPR inflections were observed that appear to report the interaction between nitrogenase and a bound substrate or a transient intermediate produced during the reduction of SCN- and CS2.