Oxygen intermediates and mixed valence states of cytochrome oxidase: infrared absorption difference spectra of compounds A, B, and C of cytochrome oxidase and oxygen.

Abstract

A study of the near-infrared absorption spectra of three oxygen compounds of membrane-bound cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase; EC 1.9.3.1) shows that the formation of compound A (oxycytochrome oxidase) causes no significant infrared absorbance changes at -1030. At -640, the formation of compound C from the mixed-valence state of the oxidase leads to increased absorption at [740][741][742][743][744][745][746][747][748][749][750] Three compounds-A, B, and C-are formed in the reaction of cytochrome oxidase with oxygen at significant concentrations at very low temperatures. They form at rates that can be extrapolated with consistency to agree with the rates of electron transfer in cytochrome oxidase as measured at room temperature in the solubilized (1, 2) and membrane-bound (3, 4) enzyme. Thus, compounds A and B appear to be functional intermediates in the oxidase reaction and seem to differ significantly from the various "oxygenated" species of cytochrome oxidase found in the presence of dithionite and oxygen (5,6) and not observed at low temperatures.The most unusual, and in many ways the most interesting, compound is C (3, 4), formed from the partially oxidized or mixed valence state oxidase (7,8) in which cytochrome a and its associated copper (9) are oxidized prior to reaction with oxygen. Cytochrome a3 reduced (and bound to CO) and its associated copper atom is presumed to remain reduced as well (7,8 This communication describes the unique infrared absorption spectra of compound C in relation to those of compounds A and B, and proposes a new structure for compound C. METHODS A split-beam, split photolysis spectrophotometer (10) was adapted to precise scanning of the infrared region of the cytochrome oxidase spectrum from 700 to 900 nm. Baseline errors in this spectral region were recorded in the digital memory. The cuvette was then illuminated on half its area to cause photolysis of the a32+-CO compound and initiation of the reaction with oxygen in the illuminated portion of the sample; the unilluminated portion still contained a32+.CO. The computer memory corrected for the baseline errors and gave the corrected difference spectrum between the two portions of the sample.Since frozen samples were usually used, no septum between the two parts was needed. No nonspecific light-scattering change was observed in the frozen sample held at constant temperature.Frozen-thawed beef heart mitochondria prepared according to the method of L6w and Vallin (11) (kindly donated by C. P. Lee) were suspended in 30% ethylene glycol (freezing point, -22°), 0.1 M mannitol, and 5 mM Tris-sulfate buffer, pH 7.4. The mitochondria, at approximately 30 mg of protein per ml, supplemented with 5 mM glutamate and 5 mM succinate, were saturated with CO, supplemented with 30% ethylene glycol by volume, resaturated with CO, and maintained at 00 [or, for longer storage, at -200, at which the preparation is stable for at least a week (12)]. Electron transport from succinate to oxygen is inhibited by about ...

The mechanism of reaction of ferricyanide-pretreated mixed-valence-state membrane-bound cytochrome oxidase with oxygen at 173 K

“…-(> 10 %) and systematic errors in the distribution of residuals. This indicated the presence of other intermediates in the reaction offerricyanide-pretreated mixed-valencestate membrane-bound cytochrome oxidase with 02 that had not been identified by low-temperature trapping and wavelength-scanning optical spectroscopy (Chance et al, 1975b,c;Chance & Leigh, 1977;Denis & Chance, 1977). A number of other models were then tested involving more intermediates and/or branching pathways, and, as in the case of the Vol.…”

“…data on intermediates IM and IIIM (Chance et al, 1975b,c;Chance & Leigh, 1977;Denis & Chance, 1977 (Chance & Leigh, 1977), where ferricyanide is the active oxidant of haem a and its reduction to any significant extent would seem unlikely. Tables 2 and 4 show that, within the errors specified, for the 444nm traces: Table 2) and the correlation coefficient (r) relating the forward and backward rate constants by using the formula (y/i2-2rV1g yv+ y/j2) , where V/1 and egj are the S.D.,n of the forward and backward rate constants.…”

“…(Palmer et al, 1976;Thomson et al, 1976Thomson et al, , 1977Falk et al, 1977), it seems likely that one of the unpaired electrons on high-spin haem a33+ could also undergo spin-couplingwith the unpaired electronO2-resulting in the formation of an exchange-coupled complex of even spin (S = 2 or 3), which would be difficult or impossible to observe by e.p.r. (b) The intense absorption band at 606-609nm associated with intermediate IlIM (Chance et al, 1975b,c;Chance & Leigh, 1977;Denis & Chance, 1977 (d) The 655nm band, which, on the basis of e.p.r. (Beinert et al, 1976) and m.c.d.…”

“…(Beinert et al, 1976) and m.c.d. (Palmer et al, 1976) studies, is thought to arise as a consequence of anti-ferromagnetic coupling between high-spin haem a33+ and CUB2+, is not formed during the course of the reaction (Chance & Leigh, 1977;Denis & Chance, 1977).…”

“…This permits one to obtain further insight into the reaction of cytochrome oxidase with 02 and the interactions of its four metal centres. Two spectroscopically distinct species, A2 and C, have been identified by using the lowtemperature trapping technique (Chance et al, 1975b,c;Denis & Chance, 1977;Chance & Leigh, 1977). Compound A2 had a 590nm ac-band with a shoulder at 600nm and a trough at 612nm, a 546nm fl-band and a negative 444nm y-band with a small 423nm peak.…”