Subunit Dissociation and Protein Unfolding in the Bovine Heart Cytochrome Oxidase Complex Induced by Guanidine Hydrochloride

Bruce C. Hill, Kim Cook, Neal C. Robinson

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23 Scopus citations


The response of cytochrome oxidase to the denaturant guanidine hydrochloride (Gdn.HCl) occurs in two stages. The first stage is a sharp transition centered at 1 M Gdn.HCl, whereas the second stage occurs from 3 to 7 M Gdn•HCl. In the first phase, changes occur in several spectroscopic properties: (1) the tryptophan fluorescence increases from 37% of that of N-acetyltryptophanamide to 85%; (2) the emission maximum shifts from 328 to 333 nm; (3) the circular dichroism (CD) signal at 222 nm diminishes by 30%; and (4) the Soret CD signal at 426 nm is completely abolished. These spectroscopic changes are accompanied by complete loss of the oxidase's steady-state electron-transfer activity. of the 13 available sulfhydryl residues, 2 are reactive in the isolated enzyme, but this number increases to almost 10 in the first stage of denaturation. Subunits III, VIb, Vic, and VII dissociate from the protein complex at 0.5 M Gdn-HCl, but only subunit VII can be recovered after gel filtration chromatography [nomenclature according to Buse et al. (1985)]. In 2.5 M Gdn-HCl, the heme groups are found with a complex consisting predominantly of subunits I, II, and IV. In the second phase of denaturation, there is further disruption in the structure of the oxidase as indicated by continued decline in the ultraviolet CD signal and shift to longer wavelength of the tryptophan emission spectrum. However, the fluorescence quantum yield and number of reactive sulfhydryl groups decrease as the denaturant level is raised. Gel filtration chromatography reveals that protein and heme form a high molecular weight aggregate at 5 M Gdn•HCl. These data indicate that cytochrome oxidase contains two regions of differing sensitivity to Gdn-HCl. The region of highest sensitivity, most likely the C domain, appears to be the subunits associated with the heme prosthetic groups. The regions most resistant to denaturation are probably the membrane-embedded M1and M2domains.

Original languageEnglish (US)
Pages (from-to)4741-4747
Number of pages7
Issue number13
StatePublished - Jun 1 1988

ASJC Scopus subject areas

  • Biochemistry


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