The nature and functional significance of the FeCu binuclear centre in cytochrome c oxidase

Abstract Cytochrome c oxidase catalyses the overall reaction shown below 4c2+ + 4H + O2 → 2H2O + 4c3+ where c2+ and c3+ are, respectively, the reduced and oxidised forms of cytochrome c. The enzyme contains four prosthetic groups; two haem iron moieties and two copper atoms [1] and a variety of spectroscopic studies have led to the conclusion that two of the four metals, one iron and one copper are involved in a substantial magnetic interaction [2]. This binuclear centre includes the haem of cytochrome a3 and one of the coppers, known variously as undetectable copper; Cuu, CuB or Cua3. A shared ligand is thought [3] to mediate the magnetic interaction and it has long been known that cythochrome a3 is the site interaction with the respiratory poisons CO and CN− and, by analogy, the substrate molecule, O2. The nature of the binuclear centre, its coupling and functional significance is clearly fundamental importance and we have attempted to explore this through the use of a range of spectroscopic approaches, both static and kinetic. Examples of ligand occupancy at each metal site in the binuclear complex are available. Carbon monoxide forms a well characterized complex with ferrous a3 [4] and has been suggested to bind also to copperIa3 [5]. Nitric oxide binds to copperIIa3 and reacts with ferric a3 as a reductant and ligand, behaving in this respect like carbon monoxide, to produce a mixed valence ligated complex of the enzyme. Cyanide reacts with both the oxidised and reduced iron of a3 blocking, in the former complex, any subsequent reduction of the a3 iron [4]. The low temperature M.C.D. spectrum between 700 and 2000 nm of the oxidised cyanide bound enzyme shows three features at 790 nm, 1564 nm and 1946 nm and the magnetic saturation curves for each of these suggests that they arise from three distinct paramagnetic centres. The features at 790 nm and 1564 nm are due to the E.P.R. detectable components of the enzyme Cua and haem a respectively. The peak at 1946 nm has the shape expected for the porphyrin → FeIII charge transfer band of a low spin ferric haem and we consider this strong evidence for its identification as haem a3-CN, although the extremely long wavelength of the peak of this band makes it unique among the near I.R. spectra of low spin ferric haems so far reported. The magnetization characteristics suggest a ground state for haem a3-CN of S = 1 and taken together with the shape indicate that this haem, in the cyanide inhibited enzyme is low spin ferric and ferromagnetically coupled to CuIIa3 to give an effective ground state spin S = 1. Treatment of the enzyme with dithionite results in the formation of a partially reduced state in which all the metal centres except a3-CN are reduced and diamagnetic. The haem a3-CN becomes E.P.R. detectable with a resonance at g = 3.58 and the 1946 nm band is lost, to be replaced at 1500 nm by a typical low spin ferric CT band whose magnetization characteristics originate from a paramagnet with gz = 3.58. This result taken together with the observations on the fully oxidised cyanide inhibited enzyme can be explained if CN− forms a bridge between haem a3 and CuIIa3, thus: FeIIICNCuIIa3. We have further investigated this species using the mixed ligand complex formed by fully oxidised cytochrome oxidase in the presence of cyanide and nitric oxide. Fully oxidised cyanide bound cytochrome oxidase reacts with NO to give a species with a g value at 3.44 in addition to 3.03 (ferric haem a) and 2.0 (copper a). On illumination at 10 K, the g = 3.44 signal is abolished but returns on warming to 50 K, behaviour not dissimilar to that of the NO complex of the fully reduced enzyme. The g= 3.44 signal is not due to partial reduction of the enzyme since the g value arising from partially reduced cyanide bound enzyme is at 3.58 and this species is not photosensitive. Low temperature M.C.D. spectroscopy shows three transitions 790 nmr (copperIIa) which disappears on illumination at 10 K to be replaced by the characteristics band at 1946 nm assigned to low spin ferric a3 CN coupled to copperIIa3. The M.C.D. saturation curved obtained at 1790 nm identifies this feature with the g = 3.44 feature seen in E.P.R. and suggests that the 1790 nm band represents haem a3+3CN influenced by the NO bound coppera3 (Cu+NO+) through the coupling which persists in this particular derivative of the enzyme. The reaction of mixed valence enzyme i.e of the reduced binuclear centre with oxygen has been followed using flow flash photolytic procedures and provides evidence at room temperature for the occurrence of Cpd A (oxyferro species) only previously seen at very low temperatures.