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The coordination environment of the Cu(B) center of the quinol oxidase from Acidianus ambivalens, a type B heme-copper oxygen reductase, was investigated by Fourier transform (FT) IR and extended X-ray absorption fine structure (EXAFS) spectroscopy. The comparative structural chemistry of dinuclear Fe-Cu sites of the different types of oxygen reductases is of great interest. Fully reduced A. ambivalens quinol oxidase binds CO at the heme a (3) center, with nu(CO)=1,973 cm(-1). On photolysis, the CO migrated to the Cu(B) center, forming a Cu (B) (I) -CO complex with nu(CO)=2,047 cm(-1). Raising the temperature of the samples to 25 degrees C did not result in a total loss of signal in the FTIR difference spectrum although the intensity of these signals was reduced sevenfold. This observation is consistent with a large energy barrier against the geminate rebinding of CO to the heme iron from Cu(B), a restricted limited access at the active-site pocket for a second binding, and a kinetically stable Cu(B)-CO complex in A. ambivalens aa (3). The Cu(B) center was probed in a number of different states using EXAFS spectroscopy. The oxidized state was best simulated by three histidines and a solvent O scatterer. On reduction, the site became three-coordinate, but in contrast to the bo (3) enzyme, there was no evidence for heterogeneity of binding of the coordinated histidines. The Cu(B) centers in both the oxidized and the reduced enzymes also appeared to contain substoichiometric amounts (0.2 mol equiv) of nonlabile chloride ion. EXAFS data of the reduced carbonylated enzyme showed no difference between dark and photolyzed forms. The spectra could be well fit by 2.5 imidazoles, 0.5 Cl(-) and 0.5 CO ligands. This arrangement of scatterers would be consistent with about half the sites remaining as unligated Cu(his)(3) and half being converted to Cu(his)(2)Cl(-)CO, a 50/50 ratio of Cu(his)(2)Cl(-) and Cu(his)(3)CO, or some combination of these formulations.