The redox reactions of the cytochrome c oxidase from Puracoccus denitrificans were investigated in a thin-layer cell designed for the combination of electrochemistry under anaerobic conditions with UVNIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surfacemodified electrode for all cofactors as indicated by the optical signals in the 400-700 nm range. Fourier transform infrared (FTIR) difference spectra of reduction and oxidation (reducedminus-oxidized and oxidized-minus-reduced, respectively) obtained in the 1800-1000 cm-' range reveal highly structured band features with major contributions in the amide 1(1620-1680 cm-') and amide II (1580-1520 cm-') range which indicate structural rearrangements in the cofactor vicinity. However, the small amplitude of the IR difference signals indicates that these conformational changes are small and affect only individual peptide groups. In the spectral region above 1700 cm-', a positive peak in the reduced state (1733 cm-') and negative peak in the oxidized state (1745 cm-') are characteristic for the formation and decay of a COOH mode upon reduction. The most obvious interpretation of this difference signal is proton uptake by one Asp or Glu side chain carboxyl group in the reduced state and deprotonation of another Asp or Glu residue. Moreover, both residues could well be coupled as a donor-acceptor pair in the proton transfer chain. An alternative interpretation is in terms of a protonated carboxyl group which shifts to a different environment in the reduced state. The relevance of this first direct observation of protein protonation changes in the cytochrome c oxidase for vectorial proton transfer and the catalytic reaction is discussed.

The thermoacidophilic archaeon Sulfolobus acidocaldarius expresses a very unusual quinol oxidase, which contains four heme a redox centers and one copper atom. The enzyme was solubilized with dodecyl maltoside and purified to homogeneity by a combination of hydrophobic interaction and anion exchange chromatography. The oxidase complex consists of four polypeptide subunits with apparent molecular masses of 64, 39, 27, and 14 kDa that are encoded by the soxABCD operon (Lü bben, M., Kolmerer, B., and Saraste, M. (1992) EMBO J. 11, 805-812). The optical spectra and redox potentials of the SoxABCD complex have been characterized, and the absorption coefficients of the contributing cytochromes a 587 and aa 3 were determined. The EPR spectra indicate the presence of three low spin and one high spin heme species, the latter associated with the binuclear heme Cu B site. Standard midpoint potentials of the cytochrome a 587 heme centers were determined as ؉210 and ؉270 mV, respectively. The maximum turnover of the complex (1300 s ؊1 at 65°C) was found to be about three times greater than that of the previously studied isolated cytochrome aa 3 subunit alone (Gleissner, ؉ /e ؊ ratio >1 was determined, identifying the SoxABCD complex as a proton-pumping quinol oxidase. According to structural analysis, the cytochrome aa 3 moiety of the complex does not contain the signature of a H ؉ pumping channel as identified in Rhodobacter sphaeroides or Paracoccus denitrificans. Therefore, for H ؉ translocation, a mechanism different from that in typical heme-copper oxidases of the aa 3 or bo 3 type is discussed.M

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.

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