Yau-Huei Wei

Ubisemiquinone radicals from the cytochrome b−c1 complex of the mitochondrial electron transport chain—Demonstration of QP-S radical formation

Abstract Stable ubisemiquinone radical(s) in the cytochrome b − c 1 -II complex of bovine heart was observed following reduction by succinate in the presence of catalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Q2. The radical showed an EPR signal of g = 2.0046 ± .003 at X band (∼9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 ± .5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved g -anisotropy and had a field separation between derivative extrema of 26 ± 1 Gauss. This radical is evidently from QP-C. These observations substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome b - c 1 -II complex only in the presence of more than a catalytic amount of succinate dehydrogenase and cytochrome b - c 1 . This signal was not antimycin a inhibitory. The signal amplitude paralleled the reconstitutive enzymic activity of succinate-cytochrome c reductase from succinate dehydrogenase and the cytochrome b - c 1 -II complex.

Electron nuclear double resonance of cytochrome oxidase: Nitrogen and proton endor from the ‘copper’ EPR signal

Proton ENDOR resonances have been found from at least two different protons with fairly large and isotropic couplings of about 12 and 19 MHz. It is possible that such protons are attached to carbons that are one bond removed from the point of ligation to copper. A number of weakly coupled protons with anisotropic couplings have also been seen. None of the protons, either weakly or strongly coupled, appears to exchange with 2H2O. We have obtained nitrogen ENDOR from at least one nitrogen with a hyperfine coupling large enough for the nitrogen to be a ligand of copper. We have not yet demonstrated experimentally ENDOR characteristic of the copper nucleus itself.

Amino acid sequence of the heme a subunit of bovine heart cytochrome oxidase and sequence homology with hemoglobin.

Abstract The amino acid sequence of the 11.6 K dalton heme a subunit of bovine heart cytochrome oxidase has been completed and is presented here. The sequence investigation has established the positions in the protein of all the possible heme ligands, namely cysteine, methionine, histidine and lysine residues. However, the isolation conditions may have caused the heme a to migrate from its original site or the heme is caged by peptides as pointed out in Reference 6. The sequence of the heme a subunit and the β-chain of hemoglobin shows homology. It is possible that these two proteins have arisen from a common ancestor in the distant past.

Amino acid sequence of bovine heart cytochrome oxidase subunit IV (Albany)

The preliminary data on the amino acid sequence of subunit IV from bovine heart cytochrome oxidase (Albany) is presented. The subunit consists of 97 amino acids linked together in a single polypeptide chain. The sequence was established by the isolation, purification and sequencing of some of the tryptic, chymotryptic and thermolytic and Staphylococcus aureus protease peptides. This subunit is present in all cytochrome oxidase preparations. It corresponds to polypeptide VIa in cytochrome oxidase (Aachen) and subunit a in cytochrome oxidase (Eugene).

The effect of phospholipid depletion on the EPR behavior of cytochrome oxidase

Abstract Phospholipids are essential components for electron transport activity of cytochrome oxidase. Recently, we have found that the removal of phospholipids from the oxidase affected the copper and low-spin heme signals, and conceivably other paramagnetic centers as demonstrated by EPR spectroscopy. At 4.2–30 °K, the signal amplitudes and power saturation behaviors were studied at approximately g = 2.0 for the copper signal, and in the neighborhood of g = 3.0 for the low-spin heme signal. After depletion of phospholipids the amplitude of the copper signal decreased 25–30% at 12–30 °K and below 12 °K 40–50% under nonsaturating conditions. The amplitude of the low-spin heme signal decreased 60–70% at 4.2–20 °K. Below 14 °K both signals became more resistant to power saturation, but the copper signal was more readily saturated above this temperature, compared to the oxidase with about 25% lipid. After removal of phospholipids, the spectral features of the copper signal remained essentially the same, but the low-spin heme signal broadened and became very asymmetric to show two signals as revealed by the second harmonic EPR spectra. These findings may explain, at least partially, the wide variations in percentage of EPR detectable copper and heme of cytochrome oxidase reported by different laboratories. Unequivocally, the EPR behavior of cytochrome oxidase is not only affected by the protein moiety, but also by the associated phospholipids of the enzyme.

EPR-detected interaction between cytochrome a3 and cytochrome a in cytochrome c oxidase

Abstract A prevailing view of cytochrome c -oxidases mode of action is that the cytochrome a moiety of the oxidase is first reduced by electrons from ferrocytochrome c , and then electron transfer occurs from cytochrome a to cytochrome a 3 and finally to oxygen [1]. We address the question of how this functional interaction is related to structural perturbation of the a 3 center by the a center or to simple proximity of the two centers. We have investigated this problem with NO complexed to heme a 3 in fully reduced oxidase ( a 2+ · Cu 1+ a · a 2+ 3 · Cu 1+ a3 ) and with NO and CO complexed to heme a 3 in a mixed valence (MV) state oxidase ( a 3+ · Cu 2+ a · a 2+ 3 · Cu 1+ a3 ). Bovine cardiac cytochrome c oxidase was prepared by methods of Refs. 2 and 3. Fully reduced and mixed valence complexes were prepared in a modified Thunberg cell under strictly anaerobic conditions. EPR (electron paramagnetic resonance) spectra were recorded under non-saturating microwave powers with a Bruker ER 420 (9.0 - 9.8 GHz) spectrometer over a 12–77 temperature range. The EPR of nitrosylferrocytochrome a 3 ( i.e. , a 3 NO in both fully reduced and MV forms showed detailed hyperfine structure from nitrogen nuclei of NO and proximal histidine. The EPR features encompassed a g-value range of g x , g z , g y = 2.09, 2.006, 1.98. The MV- a 3 -NO form, but not the fully reduced, showed additional temperature-dependent spectral changes setting in below 40 K. The most striking change occurred near g = 2.09 and smaller line-broadening changes occurred near g = 2.00. In MV- a 3 -NO there thus appears to be an internal magnetic interaction that shows rapid, temperature-dependent fluctuations down to 40 K but below 40 K slows sufficiently to allow resolution by EPR. This internal interaction has a magnitude of 12, 1.5, and 5 Gauss at g x , g z and g y respectively. At present we have two separate explanations for the phenomenon which both indicate a - a 3 interaction. Either, as a result of redox changes at cytochrome a , a changed ligand-binding environment, which has conformations that rapidly convert down to 40 K, occurs at NO-liganded cytochrome a 3 . Or, a dipolar spin-spin interaction occurs between the No-liganded and a paramagnetic metal center in cytochrome a ; this spin-spin interaction is modulated by temperature-dependent spin-lattice relaxation (which we have independently measured) of the center in cytochrome a . If the latter explanation is correct, the distance between a 3 -NO and the interacting center in cytochrome a can be estimated at 15 A, and the more likely interacting center in cytochrome a predicted to be heme a .