Determination of the oxygen affinities of terminal oxidases in Azotobacter vinelandii using the deoxygenation of oxyleghaemoglobin and oxymyoglobin: cytochrome bd is a low-affinity oxidase

Abstract

Azotobacter vinelandii is an obligately aerobic diazotrophic bacterium with two known terminal oxidases of the cytochrome o-and bd-types. The latter is required for respiratory protection of the oxygen-labile nitrogenase during aerotolerant nitrogen fixation. The apparent affinities (K,) for oxygen uptake by A. vinelandii cells and membranes respiring DL-malate have been determined by using the deoxygenation of oxyleghaemoglobin or oxymyoglobin as sensitive reporters of dissolved oxygen concentration. Dualwavelength spectrophotometery allowed continuous recording of oxygen consumption over the range 0-003-10 pM, 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 bd-type oxidase. The deoxygenation kinetics of oxyleghaemoglobin revealed a high affinity oxidase in all three strains with Km values for membrane preparations of 0013-0919 pM. In strains having the cytochrome bd-type oxidase, the Km 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 bd, perhaps due to very fast oxygen binding or scavenging by cytochrome d , 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 Km values of about 033 and 4.5 pM. The 4.5 pM component is attributed to the cytochrome bd-type oxidase because it was lacking in intact cells and membranes of the cytochrome bd-deficient mutant strain. The other two components (one with a mean Km value of about 033 pM 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 bdterminated branch of the respiratory chain and the much higher affinities for oxygen reported for the cytochrome bd-type oxidase in other bacteria.

The cioAB genes from Pseudomonas aeruginosa code for a novel cyanide‐insensitive terminal oxidase related to the cytochrome bd quinol oxidases

“…In E. coli the induction of cytochrome bd occurs at low O 2 levels that are consistent with its high-oxygen affinity. In contrast, A. vinelandii induces cytochrome bd at high oxygen levels consistent with its lower oxygen affinity and a role in protective respiration under N 2 -fixing conditions (D'Mello et al, 1994;Moshiri et al, 1991b;Kelly et al, 1990). It is clear from our data that membranes of wild-type P. aeruginosa do not contain a spectrally detectable cytochrome d signal under high-or low-aeration conditions.…”

“…The aerobic diazotroph Azotobacter vinelandii contains a spectrally similar cytochrome bd oxidase whose protein subunits show homology to E. coli CydA and CydB (Green et al, 1988;Moshiri et al, 1991a). However, the A. vinelandii cytochrome bd has a much lower O 2 affinity than the E. coli cytochrome bd (K m = 4.5 M;D'Mello et al, 1994), its expression increases at high O 2 tensions and it has a role in respiratory protection of nitrogenase (Kelly et al, 1990). A survey of bacteria that can perform cyanideinsensitive respiration (defined as respiration in the presence of 1 mM KCN) found that many bacteria contained spectrally detectable cytochrome bd (Akimenko and Trutko, 1984).…”

Regulation of expression of the cyanide-insensitive terminal oxidase in Pseudomonas aeruginosa

“…This was achieved by using identical 250 ml flasks, shaking at 200 r.p.m., but altering the medium volume. The k L a values for medium volumes of 25 ml (high), 75 ml (medium), and 150 ml (low) were estimated by the sulphite oxidation method (Pirt, 1975;Ruchti et al, 1985;Gil et al, 1992;D'Mello et al, 1994). Fig.…”

Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences

“…A protective effect against oxygen toxicity was shown for the branched respiratory system in the bacterium Azotobacter¨inelandii, which possesses an extremely oxygen sensitive nitrogenase. In this case cytochrome d is used to deplete oxygen in Ž the vicinity of the enzyme Jones, 1977;Gottschalk, 1986;D'Mello et al, 1994;Bertsova . et al, 1997 …”