No abstract

Beef heart cytochrome c oxidase has been resolved into seven subunits by electrophoresis in highly cross-linked gels containing urea and sodium dodecyl sulfate. The molecular weights of the polypeptides are estimated to be I, 35 400; II, 24 100; III, 21 000; IV, 16 800; V, 12 400; VI, 8200; and VII, 4400. It has been shown that subunits II and III can coelectrophorese on standard sodium dodecyl sulfate-polyacrylamide gels and appear as a single component with an apparent molecular weight of 22 500. This accounts for previous reports that the beef heart enzyme contains only six subunits. Amino acid analysis of the isolated subunits I, II, and III revealed that they have polarities of 35.5, 44.7, and 39.9%, respectively. All three subunits have an extremely high leucine content and a low percentage of basic amino acids relative to subunits IV-VII. The size, number, and properties of subunits in the beef heart cytochrome c oxidase complex suggest that it has essentially the same subunit structure as the complexes isolated from Saccharomyces cerevisiae and Neurospora crassa.

Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.

The rate of exchange of the labile hydrogens of lysozyme was measured by out-exchange of tritium from the protein in solution and from powder samples of varied hydration level, for pH 2, 3, 5, 7, and 10 at 25 degrees C. The dependence of exchange of powder samples on the level of hydration was the same for all pHs. Exchange increased strongly with increased hydration until reaching a rate of exchange that is constant above 0.15 g of H2O/g of protein (120 mol of H2O/mol of protein). This hydration level corresponds to coverage of less than half the protein surface with a monolayer of water. No additional hydrogen exchange was observed for protein powders with higher water content. Considered in conjunction with other lysozyme hydration data [Rupley, J. A., Gratton, E., & Careri, G. (1983) Trends Biochem. Sci. (Pers. Ed.) 8, 18-22], this observation indicates that internal protein dynamics are not strongly coupled to surface properties. The use of powder samples offers control of water activity through regulation of water vapor pressure. The dependence of the exchange rate on water activity was about fourth order. The order was pH independent and was constant from 114 to 8 mol of hydrogen remaining unexchanged/mol of lysozyme. These results indicate that the rate-determining step for protein hydrogen exchange is similar for all backbone amides and involves few water molecules. Powder samples were hydrated either by isopiestic equilibration, with a half-time for hydration of about 1 h, or by addition of solvent to rapidly reach final hydration. Samples hydrated slowly by isopiestic equilibration exhibited more exchange than was observed for samples of the same water content that had been hydrated rapidly by solvent addition. This difference can be explained by salt and pH effects on the nearly dry protein. Such effects would be expected to contribute more strongly during the isopiestic equilibration process. Solution hydrogen exchange measurements made for comparison with the powder measurements are in good agreement with published data. Rank order was proven the same for all pHs by solution pH jump experiments. The effect of ionic strength on hydrogen exchange was examined at pH 2 and pH 5 for protein solutions containing up to 1.0 M added salt. The influence of ionic strength was similar for both pHs and was complex in that the rate increased, but not monotonically, with increased ionic strength.

No abstract