Purification of Vibrio fischeri nitrite reductase and its characterization as a hexaheme c-type cytochrome.
Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of Mr 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced alpha-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.
Liu
M C
MC
Department of Chemistry, University of Oklahoma, Norman 73019.
Bakel
B W
BW
Liu
M Y
MY
Dao
T N
TN
D000596:Amino Acids / Q000032:analysis; D003574:Cytochrome c Group / Q000378:metabolism*; D006418:Heme / Q000032:analysis; D046911:Macromolecular Substances; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D009572:Nitrite Reductases / Q000302:isolation & purification*; D013053:Spectrophotometry; D014733:Vibrio / Q000201:enzymology*
D000596:Amino Acids; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D006418:Heme; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases