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 所谓的反硝化需要利用分支 电子传递链 硝酸盐被还原成亚硝酸盐， 还原醌给b型细胞色素的电子 亚基，并由此到铁硫亚基， 硝酸还原酶是一种复杂的膜结合酶， 在所有已知的净化器中。 亚硝酸盐被还原成硝酸盐 氧化物通过电子从细胞色素bc 1复合物传递到 细胞色素c551，然后到细胞色素cd 1，亚硝酸盐 还原酶在90%的分离菌株中是保守的。 的 其它的表面活性剂利用铜蛋白 将亚硝酸盐还原为一氧化氮的亚硝酸盐还原酶， 在几个测试案例中， 磷酸化，但作为亚硝酸盐解毒剂的功能。 一氧化氮还原酶，作为完整的膜结合酶 据报道，只有两个或两个以上的人几乎被净化了， 实验室，似乎是一个bc型细胞色素。 二氮 氧化还原酶是一种八铜蛋白， 有几个人，在一个案例中，有一个C。 型细胞色素亚基。 辅助酶（例如，细胞色素 c '，超氧化物歧化酶和细胞色素c过氧化物酶） 显然是为了保护这种呼吸， 可能是有害的氧化剂。 研究破环无色杆菌的铜酶， 获得铜氧还蛋白和N2 O的三级结构， 还原酶，电子对接机制的模型 将开发这些铜酶之间的转移。 基因 铜酶将在E. coli和site- 将使用定向诱变， 将基因转移到缺乏 亚硝酸盐和/或一氧化二氮还原酶。 一个目标是 在体外重建整个生理电子 从吡啶核苷酸到末端氧化剂的转移链。 两种不寻常的，也许是独一无二的细胞色素， 反硝化的辅助作用将被研究 强烈地。 这些是，第一，高自旋血红素轴承 来自B的“b '型”蛋白。可逆转的嗜盐蛋白酶 结合一氧化氮;第二，细胞色素c-552， 厌氧但非需氧生长的施氏毕赤酵母 菌株，其经历改变以成为细胞色素c 过氧化物酶。 研究的不寻常的性质， B。嗜盐菌亚硝酸盐还原酶将进行 以确定酶如何适应革兰氏阳性细胞。 的 调节D. 脱硫剂 六血红素作为亚硝酸盐或一氧化氮还原酶， 研究了

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.