Proton electron nuclear double resonance (ENDOR) spectra from the iron-molybdenum cofactor (FeMoco) of Klebsiella pneumoniae nitrogenase bound to the enzyme show that a wide variety of substrates and inhibitors, including dinitrogen, acetylene and cyanide, do not bind at or close to FeMoco in the dithionite-reduced state of the free MoFe protein, in agreement with our previous kinetic studies. Therefore models for substrate binding to FeMoco must consider structures at a more reduced level than that described by Kim and Rees [(1992) Science 257, 1677-1682]. After the enzyme has turned over in the presence of 2H2O, an additional set of protons are potentially available for exchange, namely those that can give rise to dihydrogen during enzyme turnover or generate the hydridic dinitrogen binding site; such exchangeable protons were not observed. They cannot therefore be proposed in order to explain the unusual geometry of the ‘trigonal iron atoms’ observed in the structure of FeMoco.

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