1887

Abstract

is an obligately aerobic diazotrophic bacterium with two known terminal oxidases of the cytochrome and -types. The latter is required for respiratory protection of the oxygen-labile nitrogenase during aerotolerant nitrogen fixation. The apparent affinities ( ) for oxygen uptake by cells and membranes respiring dl-malate have been determined by using the deoxygenation of oxyleghaemoglobin or oxymyoglobin as sensitive reporters of dissolved oxygen concentration. Dual-wavelength spectrophotometery allowed continuous recording of oxygen consumption over the range 0·003–10 μM, and revealed three distinct affinities for oxygen in a wild-type strain. The kinetic properties of each oxidase were distinguished by the use of two mutants, one lacking and one over-producing the cytochrome -type oxidase. The deoxygenation kinetics of oxyleghaemoglobin revealed a high affinity oxidase in all three strains with values for membrane preparations of 0·013–0·019 μM. In strains having the cytochrome -type oxidase, the values measured with intact cells were approximately fourfold higher than in membranes. These results suggest a barrier to the transfer of oxygen to the high affinity component by cytochrome , perhaps due to very fast oxygen binding or scavenging by cytochrome , or to the location of the oxygen-consuming sites of these oxidases on different faces of the membrane. The deoxygenation kinetics of oxymyoglobin revealed the presence of two components with mean values of about 0·33 and 4·5 μM. The 4·5 μM component is attributed to the cytochrome -type oxidase because it was lacking in intact cells and membranes of the cytochrome -deficient mutant strain. The other two components (one with a mean value of about 0·33 μM and the highest affinity activity) could not be assigned to particular oxidase(s). The results are interpreted in relation to the physiological role of the cytochrome -terminated branch of the respiratory chain and the much higher affinities for oxygen reported for the cytochrome -type oxidase in other bacteria.

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1994-06-01
2021-05-15
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