The active site of the bacterial nitric oxide reductase is a dinuclear iron center.
A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry. The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution. Similarly, it is dimeric in two-dimensional crystals. Images of these crystals have been processed at 8 A resolution in projection to the membrane. The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O. Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.
Hendriks
J
J
European Molecular Biology Laboratory, Heidelberg, Germany.
Warne
A
A
Gohlke
U
U
Haltia
T
T
Ludovici
C
C
Lübben
M
M
Saraste
M
M
D001665:Binding Sites; D003460:Crystallization; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D007501:Iron / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D010455:Peptides; D007501:Iron; D010088:Oxidoreductases; C034747:nitric-oxide reductase