1. Rumen fluid from four sheep, one on a low nitrogen diet, showed slight acetylene reduction.Desulfotomaculum ruminis, a rumen anaerobe, fixes N2 but the effective organisms in rumen samples seem to resembleClostridium pasteurianum; this organism can persist in the sheep rumen. In domestic sheep the contribution of rumen fixation to the animal's N-nutrition is probably negligible; other ruminants on various diets require study.
2. Respiration inAzotobacter species functions partly to protect nitrogenase from interference by oxygen. When such ‘respiratory protection’ of nitrogenase fails, the organisms reversibly ‘switch off’ nitrogenase activity, a process attributed to a change in the conformation of the nitrogenase components. When this ‘conformational protection’ fails, irreversible damage to the oxygen-sensitive protein 2 (Fe protein) of nitrogenase occurs and can be demonstrated with cell-free extracts.
3. Protein 1 (Mo-Fe protein) and protein 2 (Fe protein) ofKlebsiella pneumoniae nitrogenase, labelled with Fe57, show Mössbauer resonances tentatively assigned to ferrous and ferric iron. In mixtures, these are additive unless both ATP and Na2S2O4 (the components necessary for enzymic activity) are present, when changes take place, including the appearance of a new doublet at −0.85 and +2.2 mm/sec. Permutation of labelled and unlabelled proteins indicates that the major change occurs in protein 1. N2, C2H2, CN− or CO altered the intensity of an absorption at +2.8 mm/sec attributable to protein 2. Hence activation of the N2-ase complex involves changes in the environment of Fe but no resonances assignable to Fe-substrate binding appear.