Effects of mutation of residue I67 on redox-linked protonation processes in yeast cytochrome c oxidase.

We describe effects of a mutation, Ile-67-->Asn, in subunit I of yeast cytochrome c oxidase on redox-linked protonation processes within the protein. The mutation lowers the midpoint potential of haem a and weakens its pH dependency, but has little effect on the potential of haem a3. The residue is close to a conserved glutamate (Glu-243) in the crystal structure. We propose that protonation of Glu-243 is redox-linked to haem a, that Asn-167 perturbs its pK and that redox-linked protonation in this location is essential for the catalytic reactions of the binuclear centre. These proposals are discussed in terms of a 'glutamate trap' mechanism for proton translocation in the haem/copper oxidases.

Structural and functional analysis of deficient mutants in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae.

Four point mutations in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae that had been selected for respiratory incompetence but still contained spectrally detectable haem aa3 were analysed. The isolated mutant enzymes exhibited minor band shifts in their optical spectra and contained all eleven subunits. However, steady state activities were only a few percent compared to wild type enzyme. Using a comprehensive experimental approach, we first checked the integrity of the enzyme preparations and then identified the specific functional defect. The results are discussed using information from the recently solved structures of cytochrome c oxidase at 2.8 A. Mutation 167N is positioned between haem a and a conserved glutamate residue (E243). It caused a distortion of the EPR signal of haem a and shifted its midpoint potential by 54 mV to the negative. The high-resolution structure suggests that the primary reason for the low activity of the mutant enzyme could be that asparagine in position 67 might form a stable hydrogen bond to E243, which is part of a proposed proton channel. Cytochrome c oxidase isolated from mutant T316K did not meet our criteria for homogeneity and was therefore omitted from further analysis. Mutants G352V and V380M exhibited an impairment of electron transfer from haem a to a3 and ligand binding to the binuclear centre was affected. In mutant V380M also the midpoint potential of CuB was shifted by 65 mV to the positive. The results indicated for these two mutants changes primarily associated with the binuclear centre, possibly associated with an interference in the routes and/or sites of protonation which are required for stable formation of the catalytic intermediates. This interpretation is discussed in the light of the high resolution structure.

Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated with a novel large-scale purification method.

A novel, large-scale method for the purification of cytochrome-c oxidase from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The purified enzyme exhibited a heme a/protein ratio of 9.1 mmol/mg and revealed twelve protein bands after Tricine/SDS/PAGE. N-terminal sequencing showed that eleven of the corresponding proteins were identical to those recently described by Taanman and Capaldi [Taanman, J.-W. & Capaldi, R.A. (1992) J. Biol. Chem. 267, 22,481-22,485]. 15 of the N-terminal residues of the 12th band were identical to subunit VIII indicating that this band represents a dimer of subunit VIII (M(r) 5364). We conclude that subunit XII postulated by Taanman and Capaldi is the subunit VIII dimer and that cytochrome-c oxidase contains eleven rather than twelve subunits. We obtained the complete sequence of subunit VIa by Edman degradation. The protein contains more than 25% of charged amino acids and hydropathy analysis predicts one membrane-spanning helix. The purified enzyme had a turnover number of 1500 s-1 and the ionic-strength dependence of the Km value for cytochrome-c was similar to that described for other preparations of cytochrome-c oxidase. This was also true for the cyanide-binding characteristics of the preparation. When the enzyme was isolated in the presence of chloride, more than 90% of the preparation showed fast cyanide-binding kinetics and was resistant to formate incubation, indicating that chloride was bound to the binuclear center. When the enzyme was isolated in the absence of chloride, approximately 70% of the preparation was in the fast form. This high content of fast enzyme was also reflected in the characteristics of optical and EPR spectra for cytochrome-c oxidase purified with our method.

A protein kinase isolated from porcine brain microvessels is similar to a class of heat-shock proteins.

To further characterize a protein kinase present in porcine brain microvessels, a cDNA library using porcine microvessel poly(A) RNA was screened with polyclonal antibodies raised against the native protein kinase. Since no full-length cDNA clone could be obtained, the missing sequence information was completed using two subsequent polymerase chain reactions. The amplified transcripts were cloned and the sequence determined. Additionally, a genomic DNA library from porcine kidney was screened to substantiate the results obtained from the polymerase chain reaction. Earlier hints of a relation to a subclass of the family of heat-shock proteins (HSPs) based upon a close sequence similarity at its amino-terminus could be confirmed by comparison of the full-length cDNA sequences. Common protein kinase consensus sequences, a targeting sequence for proteins of the endoplasmic reticulum at the carboxy-terminus as well as a hydrophobic leader sequence in the amino-terminal region of the protein could also be identified. Furthermore, a set of membrane-associated substrate proteins of this enzyme could be detected in brain capillaries. The results indicate that at least some members of the HSP 90 subfamily undergo autophosphorylation and show protein kinase activity by phosphorylating substrate proteins in vitro.