Comparison of Wild-Type and nifV Mutant Molybdenum-Iron Proteins of Nitrogenase from Klebsiella pneumoniae by ENDOR Spectroscopy

Electron nuclear double resonance (ENDOR) studies of native and isotopically enriched MoFe proteins allow individual characterization of the atoms in the catalytically active FeMo-cofactor (FeMo-co) cluster of the nitrogenase MoFe protein. This paper presents ¹H, ⁵⁷Fe, ⁹⁵Mo, and ¹⁴N ENDOR measurements in a comparison of the MoFe protein isolated from wild-type Klebsiella pneumoniae (Kpl WT) and the nifV mutant of this organism (NifV^- Kpl), which contains an altered form of the FeMo-co cluster. Single-crystal-like ⁵⁷Fe ENDOR measurements show that one of the five iron sites in NifV^- Kpl has magnetic properties different from those of the wild-type protein. Proton ENDOR studies reveal an additional set of exchangeable protons that is present in NifV^- Kpl but not Kpl WT. ⁹⁵Mo ENDOR measurements show that the molybdenum site is perturbed in the cofactor. These differences are the first to be detected by physical methods and may be due to the replacement, addition, or subtraction of a non-sulfur ligand weakly bound at or near the molybdenum atom; the possible identity of such a ligand is discussed. Evidence also is presented for nitrogen coupled to the FeMo-co cluster in both proteins.