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

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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.