No abstract

Members of the cytochrome c oxidase family exhibit nitrite reductase activity. In this work, we have characterized a ferrous heme a3-nitro species in ba3-oxidase by resonance Raman spectroscopy. This provides the first evidence for the structure of a nitrite-bound species in the binuclear heme/copper center of cytochrome c oxidases.

FTIR and light-minus-dark FTIR spectroscopy have been employed to investigate the reaction of oxidized and fully reduced ba(3) oxidase with cyanide. The characterization of the structures of the bound CN(-) in the binuclear heme Fe-Cu(B) center is essential, given that a central issue in the function of ba(3) oxidase is the extent to which the partially reduced substrates interact with the two metals. In the reaction of oxidized ba(3) oxidase with cyanide the initially formed heme a(3)(3+)-C≡N-Cu(B)(2+) species with ν(CN) frequency at 2152 cm(-1) was replaced by a photolabile complex with a frequency at 2075 cm(-1) characteristic of heme a(3)(2+)-CN(-). Photolysis of the heme a(3)(2+)-CN(-) adduct produced a band at 2146 cm(-1) attributed to the formation of a transient Cu(B)(2+)-CN(-) complex. All forms are pH independent between pH 5.5-9.5 and at pD 7.5 indicating the absence of ionizable groups that influence the properties of the cyanide complexes. In contrast to previous reports, our results show that CN(-) does not bind simultaneously to both heme a(3)(2+) and Cu(B)(2+) to form the mixed valence a(3)(2+)-CN·Cu(B)(2+)CN species. The photolysis products of the heme a(3)(2+)-CN(-)/Cu(B)(2+) and heme a(3)(2+)-CN(-)/Cu(B)(1+) species are different suggesting that relaxation dynamics in the binuclear center following ligand photodissociation are dependent on the oxidation state of Cu(B).

YddV is a newly discovered signal transducer heme protein that recognizes O2 and CO. Structural differences in the ligand-bound heme complex in YddV reflect variations in catalytic regulation by O2 and CO. Time-resolved step-scan (TRS(2)) FTIR studies of the wild type and of the important in oxygen recognition and stability of the heme Fe(II)-O2 complex L65M, L65T, Y43A, Y43F and Y43W mutants were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. These mutations were designed to perturb the electrostatic field near the iron-bound gaseous ligand (CO) and also to allow us to investigate the communication pathway between the distal residues of the protein and heme. TRS(2)-FTIR spectra of YddV-heme-CO show that the heme propionates are in protonated and deprotonated states. Moreover, the rate of decay of the vibrations of amide I is on a time scale that coincides with the rate of rebinding of CO, which suggests that there is coupling between ligation dynamics in the distal heme environment and (i) relaxation of the protein backbone and (ii) the environment sensed by the heme propionates. The fast recombination rates in L65M, L65T and Y43W imply a significant role of L65 and Y43 in controlling the ligand dynamics. The implications of these results with respect to the role of the heme propionates and the charged or proton-donating residues in the distal pocket, which are crucial for stabilizing bound gaseous ligands, are discussed.

In this work we report the first spectroscopic evidence demonstrating that cbb3 oxidase catalyzes the reduction of nitrite to nitrous oxide under reducing anaerobic conditions. The reaction proceeds through the formation of a ferrous six-coordinate heme b3-nitrosyl species that has been characterized by resonance Raman spectroscopy.