Ligand Trapping by Cytochrome c Oxidase

Abstract

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe a3 3؉ -Cu B 2؉ ) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe a3 3؉ -CN-Cu B 2؉ complex. Instead the cyanide-induced transition of a preformed Fe a3 3؉ -N 3 -Cu B 2؉ to the ternary complex of Fe a3 3؉ -N 3 Cu B 2؉ -CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu B blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu B and that, during catalysis, ligands present at this copper center control access to the iron of heme a 3 from the bulk medium.

Observation of Ligand Transfer in ba3 Oxidase from Thermus thermophilus: Simultaneous FTIR Detection of Photolabile Heme a32+–CN and Transient CuB2+–CN Complexes

“…Structural information of the O 2 -binding binuclear center of ba 3 oxidase and in cytochrome c oxidase (C c O) superfamily have been determined from a great number of spectroscopic and computational studies of the CO–, CN – , and NO-bound adducts. − In general, CO binds to the ferrous (Fe 2+ ) form of heme a 3 , whereas CN – and NO bind to both oxidized (Fe 3+ ) and reduced (Fe 2+ ) forms of heme a 3 . Because of the unusual ligand-binding kinetic properties of its binuclear center, cytochrome ba 3 from T. thermophilus is unique among the heme-copper oxidases in being susceptible to a detailed analysis of its ligation dynamics in the heme a 3 -Cu B site. − Resonance Raman (RR), electron nuclear double resonance (ENDOR), and electron paramagnetic resonance (EPR) spectroscopies, in conjunction with the permutations of 13 C- and 15 N-labeled cyanide have indicated that the reaction of oxidized ba 3 with cyanide yields a heme a 3 (II)-CN·Cu B (II)–CN complex. − It has been demonstrated that cytochrome ba 3 reacts slowly with excess HCN at pH 7.4 to create a form of the enzyme in which Cu A , heme b , and Cu B remain oxidized, while heme a 3 is reduced by one electron, presumably with the formation of cyanogen .…”

“…The vibrational modes of cyanide when bound to heme-copper oxidases in various oxidation states have been studied by FTIR and RR spectroscopies. − In the oxidized form of the enzyme cyanide binds to the binuclear center to form a Fe 3+ –CN–Cu B 2+ complex in all heme-copper oxidases studied with the exception of the reports on ba 3 oxidase. − ,,,, There are some differences and conflicting reports on the fully reduced-CN complexes. In the bovine enzyme, modes at 2058 and 2045 cm –1 have been reported; in the partially reduced enzyme the modes at 2131 and 2093 cm –1 have been attributed to redox changes of the heme Fe–Cu B center or to protonation differences. , Of note, is the report of the modes observed at high CN – concentrations at 2093 and 2037 cm –1 assigned to the binding of a second CN – to Cu B .…”

Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts

“…Next, we modulated the action of the electron transport chain by the addition of N 3 − , a potent inhibitor of mitochondrial respiration and produce anoxia when bound to the metal centre of complex IV (CIV) [30] . Nonetheless, N 3 − also binds, although with less affinity, to CV [31] where it blocks ATP hydrolysis but not synthesis [32] .…”

Effect of film thickness on the structural and tribo-mechanical properties of reactive sputtered molybdenum nitride thin films

“…According to A. Chithambararaj et al [24], the bands at 2033 and 2151 cm −1 are linked to the azide bound and cyanide group (C≡N), respectively. An additional mode at 2048 cm −1 belongs to free azide, while at 2092 cm −1 belongs to free HCN [31]. The bands at 2300-2400 cm −1 are also attributed to the stretching of the C=N bond [32].…”