NITROGENASE OF KLEBSIELLA-PNEUMONIAE - ELECTRON-NUCLEAR DOUBLE-RESONANCE (ENDOR) STUDIES ON THE SUBSTRATE REDUCTION SITE

Proton electron nuclear double resonance (ENDOR) spectra iron-molybden um cofactor (FeMoco) of Klebsiella pneumoniae nitrogenase bound to the enzyme show that a wide variety of substrates and inhibitors, includi ng dinitrogen, acetylene and cyanide, do not bind at or close to FeMoc o in the dithionite-reduced state of the free MoFe protein, in agreeme nt with our previous kinetic studies. Therefore models for substrate b inding to FeMoco must consider structures at a more reduced level than that described by Kim and Bees [(1992) Science 257, 1677-1682]. After the enzyme has turned over in the presence of (H2O)-H-2, additional s et of protons are potentially available for exchange, namely those tha t can give rise to dihydrogen during enzyme turnover or generate the h ydridic dinitrogen binding site; such exchangeable protons were not ob served. They therefore be proposed in order to explain the unusual geo metry of the 'trigonal iron atoms' observed in the structure of FeMoco .