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.

Intramolecular proton transfer of heme-copper oxidases is performed via the K- and the transmembrane D-channels. A carboxyl group conserved in a subgroup of heme-copper oxidases, located within the D-channel close to the binuclear center (=glutamic acid-286 in cytochrome bo(3) from Escherichia coli) is essential for proton pumping. Upon electron transfer to the fully oxidized (FO) enzyme, this amino acid has been shown to undergo a cyanide-independent environmental change. The redox-induced environmental transition of glutamic acid-286 is preserved in the site-directed mutant Y288F, which has lost its Cu(B) binding capacity. Furthermore, the mixed-valence (MV) redox state of cytochrome bo(3) (in which Cu(B) and high-spin heme are reduced, whereas the low-spin heme stays oxidized) was prepared by anaerobic exposure of the protein to carbon monoxide. This complex was converted (i) to the FO state by reaction with the caged dioxygen donor mu-peroxo) (mu-hydroxo) bis [bis (bipyridyl) cobalt (III)] and (ii) to the fully reduced (FR) state via caged electron donors; the environmental change of glutamic acid-286 could be observed only upon reduction. Taken together, these results from two different lines of evidence clearly show that the redox transition of the low-spin heme b center alone triggers the change in the chemical environment of this acidic side chain. It is suggested that glutamic acid-286 is a kinetic enhancer of proton translocation, which is energetically favoured in mesophilic oxidases.

We developed the synthesis of the caged oxygen donor (µ‐peroxo)(µ‐hydroxo)bis[bis(bipyridyl)cobalt(III)] complex (HPBC) as nitrate salt, which has, compared with the perchlorate‐form described previously [MacArthur, R., Sucheta, A., Chong, F.F. & Einarsdottir, Ö. (1995) Proc. Natl Acad. Sci. USA, 92, 8105–8109], greatly enhanced solubility. Now, the quantum efficiency of the photolytical release of dioxygen was determined to be 0.4 per photon at a laser wavelength of 308 nm, which was used to observe biological reactions. The X‐ray structure of HPBC has been solved, and the molecular interactions of photochemically generated oxygen with cytochrome oxidase were investigated with optical and FT‐IR spectroscopy: it acts as acceptor of electrons transferred from prereduced cytochrome bo3, the heme‐copper oxidase from Escherichia coli. FT‐IR spectra revealed typical absorbance difference changes in the carbonyl region of cytochrome bo3, supported by bandshifts due to solvent isotope exchange and by assignment using site‐directed mutants. IR difference spectra of the photooxidation reaction using the caged oxygen compound, and of the photoreduction reaction using the caged electron donor FMN, have inverted shapes. The spectroscopic signals of carboxyl groups are thus equivalent in both reactions: the use of chemically produced oxygen allows the observation of the ongoing molecular changes of cytochrome bo3 oxidase under quasi‐physiological conditions.