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
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
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DOI：
10.1099/00221287-140-6-1395
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发表时间：
1994-06-01
期刊：
影响因子：
--
通讯作者：
POOLE, RK
中科院分区：
文献类型：
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作者：
DMELLO, R;HILL, S;POOLE, RK
棕色固氮菌是一种专性好氧固氮菌，具有两种已知的细胞色素o型和bd型末端氧化酶。后者是耐氧固氮过程中对氧不稳定固氮酶的呼吸保护所必需的。测定了A. vinelandii细胞和膜呼吸DL-苹果酸已经通过使用氧合血红蛋白或氧合肌红蛋白的脱氧作为溶解氧浓度的敏感报告物来确定。双波长荧光光谱仪允许连续记录的氧消耗在0.003-10 μ M的范围内，并揭示了三个不同的亲和力，在野生型菌株的氧。每个氧化酶的动力学特性的区别使用两个突变体，一个缺乏和一个过度生产的细胞色素KD型氧化酶。氧合血红蛋白的脱氧动力学揭示了在所有三种菌株中具有高亲和力的氧化酶，膜制备物的Km值为0.013-0.019 μ M。在具有细胞色素bd型氧化酶的菌株中，用完整细胞测得的Km值比在膜中高约四倍。这些结果表明，通过细胞色素kd的高亲和力组分的氧转移的障碍，可能是由于非常快的氧结合或清除细胞色素d，或这些氧化酶的耗氧位点的膜的不同面上的位置。氧合肌红蛋白的脱氧动力学揭示了两种组分的存在，平均Km值约为0.33和4.5 μ M。4.5 μ M组分归因于细胞色素bd型氧化酶，因为它缺乏细胞色素bd缺陷突变株的完整细胞和膜。其它两种组分（一种具有约0.33 μ M的平均K-m值和最高的亲和活性）不能归属于特定的氧化酶。结果被解释在呼吸链的细胞色素bd-终止的分支的生理作用和更高的亲和力为氧报告的细胞色素kd-型氧化酶在其他细菌。
Azotobacter vinelandii is an obligately aerobic diazotrophic bacterium with two known terminal oxidases of the cytochrome o- and bd-types. The latter is required far respiratory protection of the oxygen-labile nitrogenase during aerotolerant nitrogen fixation. The apparent affinities (K-m) 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. Dual-wavelength spectrophotometery allowed continuous recording of oxygen consumption over the range 0.003-10 mu M, 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 kd-type oxidase. The deoxygenation kinetics of oxyleghaemoglobin revealed a high 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 approximately fourfold higher than in membranes. These results suggest a barrier to the transfer of oxygen to the high affinity component by cytochrome kd, 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 K-m values 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 cells and membranes of the cytochrome bd-deficient mutant strain. The other two components (one with a mean K-m value of about 0.33 mu M 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 bd-terminated branch of the respiratory chain and the much higher affinities for oxygen reported for the cytochrome kd-type oxidase in other bacteria.