Effects of substrates (methyl isocyanide, C2H2) and inhibitor (CO) on resting-state wild-type and NifV(-)Klebsiella pneumoniae MoFe proteins.

We report the use of electron nuclear double resonance (ENDOR) spectroscopy to examine how the metal sites in the FeMo-cofactor cluster of the resting nitrogenase MoFe protein respond to addition of the substrates acetylene and methyl isocyanide and the inhibitor carbon monoxide. 1H, 57Fe and 95Mo ENDOR measurements were performed on the wild-type and the NifV(-)proteins from Klebsiella pneumoniae. Among the molecules tested, only the addition of acetylene to either protein induced widespread changes in the 57Fe ENDOR spectra. Acetylene also induced increases in intensity from unresolved protons in the proton ENDOR spectra. Thus we conclude that acetylene may bind to the resting-state MoFe protein to perturb the FeMo-cofactor environment. On the other hand, the present results show that methyl isocyanide and carbon monoxide do not substantially alter the FeMo cofactor's geometric and electronic structures. We interpret this as lack of interaction between those two molecules and the FeMo cofactor in the resting state MoFe protein. Thus, although it is generally accepted that substrates or inhibitors bind to the FeMo-cofactor only under turnover condition, this work provides evidence that at least one substrate can perturb the active site of nitrogenase under non-catalytic conditions.