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Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy.

ABSTRACT: Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)--CN in the partially reduced state. Hence as haem a3+(3)--CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)--CN . Cu+a3.

SUBMITTER: Johnson MK

PROVIDER: S-EPMC1162657 | biostudies-other | 1981 Mar

REPOSITORIES: biostudies-other

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Project description:The purification of formate dehydrogenase (FDH) from Pseudomonas aeruginosa after anaerobic growth on nitrate-containing medium was carried out. The separation of the FDH enzyme from nitrate reductase (NiR), which are found together in a particle fraction and constitute the short respiratory chain of this bacterium, has been followed by optical, magnetic c.d. (m.c.d.) and e.p.r. spectroscopy. These techniques have allowed the haem, iron-sulphur clusters and molybdenum components to be detected and, in part, their nature to be determined. Attempts to extract FDH anaerobically in the absence of sodium dithionite led to loss of activity. Addition of sodium dithionite maintained the activity of the enzyme, even after subsequent exposure to air, in an assay involving formate reduction with Nitro Blue Tetrazolium as reductant. Three preparations of FDH have been examined spectroscopically. The preparations vary in the amount of contaminating nitrate reductase, the amount of cytochrome c present and the concentration of oxidized [3Fe-4S] cluster. Optical spectra and low-temperature m.c.d. spectroscopy show the loss of a cytochrome-containing protohaem IX co-ordinated by methionine and histidine as NiR is separated from the preparation. In its purest state FDH contains one molecule of cytochrome co-ordinated by two histidine ligands in the oxidized state. This cytochrome has an e.p.r. spectrum with gz = 3.77, the band having the unusual ramp shape characteristic of highly anisotropic low-spin ferric haem. It also shows a charge-transfer band of high intensity in the m.c.d. spectrum at 1545 nm. It has recently been shown [Gadsby & Thomson (1986) FEBS Lett. 197, 253-257] that these spectroscopic properties are diagnostic of a bishistidine co-ordinated haem with steric constraint of the axial ligands. The e.p.r. and m.c.d. spectra of the reduced state of FDH reveal the presence of an iron-sulphur cluster of the [4Fe-4S]+ type. The g-values are 2.044, 1.943 and 1.903. An iron-sulphur cluster of the class [3Fe-4S], detected by e.p.r. spectroscopy in the oxidized state and by low-temperature m.c.d. spectroscopy in the reduced state, is purified away with the NiR. Finally, an e.p.r. signal at g = 2.0 with a narrow bandwidth which persists to 80 K is observed in the purest preparation of FDH. This may arise from an organic radical species.

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Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy.

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