DETERMINATION OF THE OXYGEN AFFINITIES OF TERMINAL OXIDASES IN AZOTOBACTER-VINELANDII USING THE DEOXYGENATION OF OXYLEGHEMOGLOBIN AND OXYMYOGLOBIN - CYTOCHROME BD IS A LOW-AFFINITY OXIDASE

Azotobacter vinelandii is an obligately aerobic diazotrophic bacterium with two known terminal oxidases of the cytochrome o- and bd-types. T he latter is required far respiratory protection of the oxygen-labile nitrogenase during aerotolerant nitrogen fixation. The apparent affini ties (K-m) for oxygen uptake by A. vinelandii cells and membranes resp iring DL-malate have been determined by using the deoxygenation of oxy leghaemoglobin or oxymyoglobin as sensitive reporters of dissolved oxy gen concentration. Dual-wavelength spectrophotometery allowed continuo us recording of oxygen consumption over the range 0.003-10 mu M, and r evealed three distinct affinities for oxygen in a wild-type strain. Th e kinetic properties of each oxidase were distinguished by the use of two mutants, one lacking and one over-producing the cytochrome kd-type oxidase. The deoxygenation kinetics of oxyleghaemoglobin revealed a h igh affinity oxidase in all three strains with K-m values for membrane preparations of 0.013-0.019 mu M. In strains having the cytochrome bd -type oxidase, the K-m values measured with intact cells were approxim ately fourfold higher than in membranes. These results suggest a barri er to the transfer of oxygen to the high affinity component by cytochr ome kd, perhaps due to very fast oxygen binding or scavenging by cytoc hrome d, or to the location of the oxygen-consuming sites of these oxi dases on different faces of the membrane. The deoxygenation kinetics o f oxymyoglobin revealed the presence of two components with mean K-m v alues of about 0.33 and 4.5 mu M. The 4.5 mu M component is attributed to the cytochrome bd-type oxidase because it was lacking in intact ce lls and membranes of the cytochrome bd-deficient mutant strain. The ot her two components (one with a mean K-m value of about 0.33 mu M and t he highest affinity activity) could not be assigned to particular oxid ase(s). The results are interpreted in relation to the physiological r ole of the cytochrome bd-terminated branch of the respiratory chain an d the much higher affinities for oxygen reported for the cytochrome kd -type oxidase in other bacteria.