The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation.

Klebsiella pneumoniae synthesized only b-type and d-type cytochromes under the wide range of growth conditions tested, and reaction with CO revealed two potential oxidases. The o-type oxidase was produced only in the presence of O2 and appeared to be repressed by glucose. The d-type oxidase was, by contrast, produced only in the absence of measurable O2 (less than 1 microM), and was the only oxidase expressed in nitrogen-fixing conditions. It was extracted from the membrane, purified and shown to be similar to that from E. coli in being a heterodimer (subunits of Mr 52,000 and 35,000), in containing two distinguishable b haems and haem d (one or two molecules per molecule of oxidase), and in being able to react with O2 to give a stable oxygenated intermediate. The purified d-type cytochrome oxidase had a very high affinity for O2 (Km 20 nM; measured by the spectral properties of leghaemoglobin). It is proposed that this provides a role for this oxidase in lowering the O2 concentration to allow nitrogenase synthesis and function, and to provide a terminal oxidase to permit electron-transport-coupled ATP synthesis which supports the increase in efficiency of nitrogen fixation observed under microaerobic conditions.