lecular Biology and Biochemistry of Heme and Copper- ntaining Dissimilatory Denitrifying Enzymes

这种类型的厌氧（或替代）细菌呼吸称为反硝化需要利用支链电子传递链。硝酸钾被还原醌给予b型细胞色素亚基的电子还原为亚硝酸盐，然后被还原为硝酸盐还原酶的钼铁硫亚基，这是一种复杂的膜结合酶，在所有已知的反硝化菌中都是保守的。亚硝酸盐通过电子从细胞色素bc1复合物到细胞色素c551再到细胞色素cd1还原为一氧化氮，在90%的反硝化分离物中亚硝酸盐还原酶是保守的。其他明显的反硝化剂利用铜蛋白亚硝酸盐还原酶将亚硝酸盐还原为一氧化氮，并且在几次测试中似乎没有将还原与磷酸化联系起来，而是作为亚硝酸盐解毒剂发挥作用。一氧化氮还原酶是一种完整的膜结合酶，据报道仅在两个或三个实验室中纯化过，似乎是一种bc型细胞色素。氧化亚氮还原酶是一种含有8个铜的蛋白质，在几种反硝化菌中保守存在，在一个案例中报道具有c型细胞色素亚基。辅助酶（如细胞色素c′、超氧化物歧化酶和细胞色素c过氧化物酶）显然可以保护这种类型的呼吸免受可能有害的氧化剂的伤害。为了研究无色杆菌的铜酶，获得铜氧还蛋白和N2O还原酶的三级结构，建立铜酶之间电子传递的对接机制模型。铜酶的基因将在大肠杆菌中克隆，位点定向诱变将被用于将基因转移到缺乏亚硝酸盐和/或氧化亚氮还原酶的stutzeri突变体中。一个目标将是在体外重建从吡啶核苷酸到末端氧化剂的整个生理电子转移链。两个不寻常的，也许是独特的，细胞色素，似乎承担辅助作用的反硝化将被深入研究。首先是高自旋血红素的“b”型蛋白质，它可以可逆地结合一氧化氮；二是来自厌氧而非好氧培养的stutzeri Zobell菌株的细胞色素c-552，该菌株经过改变成为细胞色素c过氧化物酶。研究卤代反硝化芽孢杆菌亚硝酸盐还原酶的不寻常性质，以确定该酶如何适应革兰氏细胞。研究了脱硫剂六血红素作为亚硝酸盐还原酶和一氧化氮还原酶对脱硫性能的调节机制。

That type of anaerobic (or alternative) bacterial respiration called denitrification entails utilization of branched electron transport chains. Nitrate is reduced to nitrite by electrons donated by reduced quinones to a b-type cytochrome subunit and thence to the molybdo-iron-sulfur subunit of nitrate reductase, a complex, membrane-bound enzyme conserved in all known denitrifiers. Nitrite is reduced to nitric oxide by electrons passing from the cytochrome bc1 complex to cytochrome c551 and then to cytochrome cd1, the nitrite reductase conserved in 90% of denitrifying isolates. The other apparent denitrifiers make use of a copper protein nitrite reductase that reduces nitrite to nitric oxide and appears in several cases tested not to link reduction to phosphorylation but to function as a nitrite detoxifier. Nitric oxide reductase, as integrally membrane-bound enzyme and reportedly very nearly purified in only two or there laboratories, appears to be a bc-type cytochrome. Nitrous oxide reductase is an eight-copper protein conserved in several denitrifiers and reported in one case to have a c- type cytochrome subunit. Ancillary enzymes (e.g., cytochrome c',superoxide dismutase, and cytochrome c peroxidase) apparently serve to protect this type of respiration from possibly damaging oxidants. To study the copper enzymes of Achromobacter cycloclastes, obtaining tertiary structures for cupredoxin and the N2O reductase, a Model of docking mechanisms for electron transfer among these copper enzymes will be developed. Genes for the copper enzymes will be cloned in E. coli and site- directed mutagenesis will be used with the possibility of transfer of the genes into P. stutzeri mutants lacking nitrite and/or nitrous oxide reductase. One goal will be reestablishment in vitro of the entire physiological electron transfer chain from pyridine nucleotide to terminal oxidant. Two unusual, perhaps unique, cytochromes that appear to bear ancillary roles in denitrification will be studied intensively. These are, first, the high-spin-heme-bearing "b'-type" protein from B. halodenitrificans which reversibly binds nitric oxide; and, second, the cytochrome c-552 from anaerobically but not aerobically grown P. stutzeri Zobell strain, which undergoes alteration to become a cytochrome c peroxidase. Investigation of the unusual properties of the B. haloldenitrificans nitrite reductase will be carried out to determine how the enzyme fits into the Gram + cell. The mechanism for regulating the performance of D. desulfuricans hexaheme as either nitrite or nitric oxide reductase will be investigated.