Reconstitution of iron-sulfur cluster of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1.
NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system, is an ion-sulfur flavoprotein with one FAD and one iron-sulfur cluster of [2Fe-2S] type (Yamaguchi, M., and Fujisawa, H. (1978) J. Biol. Chem. 253, 8848-8853). Treatment of NADH-cytochrome c reductase with p-chloromercuriphenylsulfonic acid resulted in fading of its color with a concomitant loss of the NADH-cytochrome c reductase activity. The p-chloromercuriphenylsulfonic acid-treated enzyme was found to contain one FAD but no significant amounts of iron and labile sulfide. Incubation of the iron-sulfur-depleted enzyme with ferrous ions and sulfide in the presence of 2-mercaptoethanol led to both reconstitution of iron-sulfur cluster of the enzyme and concomitant restoration of the enzyme activity. Although the iron-sulfur-depleted enzyme catalyzed NADH-dependent reduction of ferricyanide, nitroblue tetrazolium, or oxygen, it could not catalyze NADH-dependent reduction of the oxygenase component of benzoate 1,2-dioxygenase system. In contrast, the reconstituted enzyme recovered the activity of NADH-dependent reduction of the oxygenase component to the original level. Certain other catalytic and molecular properties of the iron-sulfur-depleted enzyme and the reconstituted enzyme are also presented.
D001565:Benzoates / Q000378:metabolism; D003579:Cytochrome Reductases / Q000378:metabolism*; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008667:Metalloproteins / Q000378:metabolism*; D009245:NADH Dehydrogenase / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013440:Sulfides / Q000032:analysis
D001565:Benzoates; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D008667:Metalloproteins; D013440:Sulfides; D005182:Flavin-Adenine Dinucleotide; D007501:Iron; D010105:Oxygenases; C019766:benzoate 1,2-dioxygenase; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase