THE ACTIVE-SITE OF THE BACTERIAL NITRIC-OXIDE REDUCTASE IS A DINUCLEAR IRON CENTER

A novel, improved method for purification of nitric oxide reductase (N OR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome be complex which, according to protei n chemical and hydrodynamic data, contains two subunits in a 1:1 stoic hiometry. 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 proces sed at 8 Angstrom resolution in projection to the membrane. The NorB s ubunit is homologous to the main catalytic subunit of cytochrome oxida se and is predicted to contain the active bimetallic center in which t wo NO molecules are turned over to N2O. Metal analysis and heme compos ition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform i nfrared spectroscopy shows that carbon monoxide dissociates from the r educed heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron param agnetic resonance spectroscopy reveals that NO binds to the reduced en zyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal wherea s the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.