Purification of cytochrome cd1 nitrite reductase from Pseudomonas stutzeri JM300 and reconstitution with native and synthetic heme d1.
Cytochrome cd1 nitrite reductase has been purified from Pseudomonas stutzeri strain JM 300. This enzyme appears to be a dimer with a subunit molecular mass of 54 kDa and its isoelectric point is determined to be 5.4. The N terminus of amino acid sequence has strong homology with that of nitrite reductase from P. aeruginosa. The apoprotein of this enzyme has been reconstituted with native and synthetic heme d1. The nitrite reductase activity measured by NO and N2O gas evolution can be restored to 82% of the activity of the original enzyme when the protein was reconstituted with the native heme d1 and to 77% of the activity when reconstituted with the synthetic heme d1. The absorption spectra of both reconstituted enzymes are essentially identical to that of the original nitrite reductase. These results further substantiate the novel dione structure of heme d1 as proposed. The loss of NO2- reducing activity in the absence of heme d1 and its restoration by addition of heme d1 provides further evidence that heme d1 plays a key role in the conversion of NO2- to NO and N2O.
Weeg-Aerssens
E
E
Department of Chemistry, Michigan State University, East Lansing 48824.
Wu
W S
WS
Ye
R W
RW
Tiedje
J M
JM
Chang
C K
CK
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003580:Cytochromes; D003576:Electron Transport Complex IV / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009569:Nitric Oxide / Q000378:metabolism; D009572:Nitrite Reductases; D009609:Nitrous Oxide / Q000378:metabolism; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D013056:Spectrophotometry, Ultraviolet
D003580:Cytochromes; D009569:Nitric Oxide; D006418:Heme; C029400:heme d1; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); D003576:Electron Transport Complex IV; D009609:Nitrous Oxide