Garland P B, Downie J A, Haddock B A
Biochem J. 1975 Dec;152(3):547-59. doi: 10.1042/bj1520547.
Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.
已对在硝酸盐存在下厌氧生长的大肠杆菌原生质球进行了与硝酸盐呼吸还原相关的质子转运化学计量和速率的测量。观察到的化学计量[导致H⁺/NO₃⁻比率；P. 米切尔（1966年）《氧化磷酸化和光合磷酸化中的化学渗透偶联》，格林研究公司，博德明]对于L - 苹果酸氧化约为4，对于琥珀酸、D - 乳酸和甘油氧化约为2。以甲酸盐作为还原剂、氧气或硝酸盐作为氧化剂来测量导致H⁺/2e⁻比率时，因与甲酸盐摄取和二氧化碳形成相关的pH变化而变得复杂。然而，可以得出结论，甲酸盐氧化位点在细胞质膜的内侧，甲酸盐氧化的导致H⁺/O比率约为4，且导致H⁺/NO₃⁻比率大于2。对硝酸盐渗透到对渗透压敏感的原生质球中的速率测量表明，硝酸盐阴离子存在电致性进入。在30℃时硝酸盐进入的渗透系数在10⁻⁹至10⁻¹⁰厘米·秒⁻¹之间。在通常用于测定硝酸盐还原酶（EC 1.7.99.4）活性的浓度下计算出的硝酸盐进入速率约为还原型苄基紫精所观察到的硝酸盐还原速率所需速率的0.1%。对一个无血红素突变体（其硝酸盐还原酶去阻遏但无法通过呼吸链还原硝酸盐）细胞内和细胞外空间之间硝酸盐分布的测量表明，无论是否添加葡萄糖，硝酸盐都不会在细胞内浓缩。渗透膨胀实验表明，与叠氮酸的快速扩散相比，叠氮阴离子穿过细胞质膜的扩散速率相对较低。叠氮化物对硝酸盐还原酶的抑制作用不会因改变细胞质膜跨膜pH梯度的处理而改变。得出的结论是，硝酸盐还原酶对叠氮化物敏感的硝酸盐还原位点位于细胞质膜的外侧。讨论了该位置对由硝酸盐还原驱动的质子转运机制的影响，并提出细胞质膜的硝酸盐还原酶是有方向性的，在膜外侧用从膜内侧穿过的2H⁺和2e⁻还原硝酸盐。
