Cytochrome aa3 of Rhodobacter sphaeroides and cytochrome bo of E. coli are useful models of the more complex cytochrome c oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the heme a3 (or o)-CuB center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.

Amyloid is a term for extracellular protein fibril deposits that have characteristic tinctorial and structural properties. Heparan sulphate, or the heparan sulphate proteoglycan perlecan, has been identified in all amyloids and implicated in the earliest stages of inflammation-associated (AA) amyloid induction. Heparan sulphate interacts with the AA amyloid precursor and the beta-peptide of Alzheimer's amyloid, imparting characteristic secondary and tertiary amyloid structural features. These observations suggest that molecules that interfere with this interaction may prevent or arrest amyloidogenesis. We synthesized low-molecular-weight (135-1,000) anionic sulphonate or sulphate compounds. When administered orally, these compounds substantially reduced murine splenic AA amyloid progression. They also interfered with heparan sulphate-stimulated beta-peptide fibril aggregation in vitro.

The cupredoxin fold, a Greek key beta‐barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C‐terminal fragment of the membrane‐bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water‐soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.

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Cytochrome bo from Escherichia coli is a ubiquinol oxidase which is a member of the superfamily of heme-copper respiratory oxidases. This superfamily, which includes the eukaryotic cytochrome c oxidases, has in common a bimetallic center consisting of a high-spin heme component and a copper atom (CuB) which is the site where molecular oxygen is reduced to water. Subunit I, which contains all the amino acid ligands to the metal components of the binuclear center, has 15 putative transmembrane spanning helices, of which 12 are common to the entire superfamily. Transmembrane helix VIII has been noted to contain highly conserved polar residues that fall along one face of the helix. These residues could, in principle, be important components of a pathway providing a conduit for protons from the cytoplasm to gain access to the binuclear center. These conserved residues include Thr352, Thr359, and Lys362. In addition, Pro358, in the middle of this transmembrane helix, is totally conserved in the superfamily. Some substitutions for Thr352 (Ala, Asn) result in major perturbations at the binuclear center as judged by the low-temperature Fourier transform infrared (FTIR) absorbance difference spectroscopy of the CO adducts. Whereas Thr352Ala is inactive enzymatically, both Thr352Asn and Thr352Ser have substantial activity. Substitutions for Thr359 (Ala or Ser) also do not perturb the spectroscopic properties of the binuclear metal center, but the Thr359Ala mutant is devoid of enzyme activity. Changing the neighboring Pro358 to Ala has no detectable effect on the properties of the oxidase. However, all substitutions for Lys362 (Leu, Met, Gln, or Arg) are inactive.(ABSTRACT TRUNCATED AT 250 WORDS)