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 Sacchar omyces cerevisiae that had been selected for respiratory incompetence but still contained spectrally detectable haem aa(3) were analysed. Th e isolated mutant enzymes exhibited minor band shifts in their optical spectra and contained all eleven subunits. However, steady state acti vities were only a few percent compared to wild type enzyme. Using a c omprehensive experimental approach, we first checked the integrity of the enzyme preparations and then identified the specific functional de fect, The results are discussed using information from the recently so lved structures of cytochrome c oxidase at 2.8 Angstrom. Mutation I67N 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 m idpoint potential by 54 mV to the negative. The high-resolution struct ure suggests that the primary reason for the low activity of the mutan t enzyme could be that asparagine in position 67 might form a stable h ydrogen bond to E243, which is part of a proposed proton channel, Cyto chrome c oxidase isolated from mutant T316K did not meet our criteria for homogeneity and was therefore omitted from further analysis. Mutan ts G352V and V380M exhibited an impairment of electron transfer from h aem a to a(3) and ligand binding to the binuclear centre was affected. In mutant V380M also the midpoint potential of Cu-B was shifted by 65 mV to the positive. The results indicated for these two mutants chang es 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 struc ture. (C) 1997 Elsevier Science B.V.