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作用，需要利用分支的电子传输链。硝酸盐被还原为亚硝酸盐，电子由还原的苯二酚提供给b型细胞色素亚基，然后传递到硝酸还原酶的钼铁硫亚基，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复合体传递到细胞色素C551，然后转化为细胞色素CD1，亚硝酸盐还原酶在90%的反硝化菌中保守。其他明显的反硝化菌利用一种铜蛋白亚硝酸盐还原酶将亚硝酸盐还原为一氧化氮，在几种情况下似乎不是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。一氧化氮合酶是一种BC型细胞色素，是一种完整的膜结合酶，据报道，只有两个或两个以上的实验室进行了纯化。一氧化二氮还原酶是一种由8个铜组成的蛋白质，存在于多种反硝化酶中，其中一例报道含有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)显然起到保护这种类型的呼吸免受可能有害的氧化剂的作用。为了研究嗜铬杆菌环状芽孢杆菌的铜酶，获得铜还蛋白和N2O还原酶的三级结构，将建立铜酶之间电子转移的对接机制模型。铜酶基因将在大肠杆菌中克隆，定点突变将用于将基因转移到缺乏亚硝酸盐和/或一氧化二氮还原酶的斯图策里突变株中。一个目标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。两种不同寻常的，也许是独特的，似乎在反硝化过程中起辅助作用的细胞色素将被深入研究。这些是，第一，从盐生亚硝酸杆菌中可逆地结合一氧化氮的高自旋含血红素的“b‘型”蛋白质；第二，来自厌氧但不是好氧生长的斯图兹里杆菌Zobell菌株的细胞色素c-552，它经历了变化成为细胞色素c过氧化物酶。将对卤代脱氮假单胞菌亚硝酸盐还原酶的不寻常性质进行研究，以确定该酶如何适应革兰氏阳性细胞。此外，还将探讨其作为亚硝酸盐或一氧化氮还原酶的作用机制。

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.