A novel microperoxidase activity: methyl viologen-linked nitrite reducing activity of microperoxidase.
To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, the fragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis of commercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its fifth ligand. The nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the cutting of the peptide chain. The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acid residues) and 3.2-fold that of nitrite reductase (EC 1.7.7.1) from Escherichia coli. In the nitrite reduction by mp, nitrite was completely reduced to ammonia. We presumed that ferrous mps reduced NO2- to NO by donating one electron, the NO was completely reduced to NH4+ under anaerobic condition via ferrous-NO complexes as a reaction intermediate using visible spectra and ESR spectra, and this overall reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized mp9 had a nitrite reducing activity similar to that of mp9 in solution, and the resin retained the activity after five uses and even 1-year storage. The mp will be able to use as a substitute for nitrite reductase.
Suruga
Kohei
K
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan.
Murakami
Kiriko
K
Taniyama
Yohei
Y
Hama
Toshiyuki
T
Chida
Hirotaka
H
Satoh
Tadashi
T
Yamada
Seiji
S
Hakamata
Wataru
W
Kawachi
Ryu
R
Isogai
Yasuhiro
Y
Nishio
Toshiyuki
T
Oku
Tadatake
T
D020106:Acrylamide / Q000737:chemistry; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000378:metabolism; D004227:Dithionite / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D009573:Nitrites / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010269:Paraquat / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D005290:Ferric Compounds; D005296:Ferrous Compounds; D009573:Nitrites; D004227:Dithionite; D020106:Acrylamide; D000641:Ammonia; D012685:Sepharose; D010544:Peroxidases; C021799:microperoxidase; D010269:Paraquat