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Biochimica et Biophysica Acta 1321 1997 79–92
Structural and functional analysis of deficient mutants in subunit I of
cytochrome c oxidase from Saccharomyces cereÕisiae
Claus Ortwein
a
, Thomas A. Link
a
, Brigitte Meunier
b,1
, Anne-Marie Colson-Corbisier
c
,
Peter R. Rich
1b
, Ulrich Brandt
a,)
a
UniÕersitatsklinikum Frankfurt, Zentrum der Biologischen Chemie, Theodor-Stern-Kai 7, Haus 25B, 60590 Frankfurt am Main,
¨
Germany
b
Glynn Research Institute, Glynn Bodmin, Cornwall PL30 4AU, United Kingdom
c
Unite de Genetique, UniÕersite Catholique de LouÕain, 1348 LouÕain la NeuÕe, Belgium
´´´ ´
Received 7 March 1997; accepted 20 March 1997
Abstract
Four point mutations in subunit I of cytochrome c oxidase from Saccharomyces cereÕisiae that had been selected for
respiratory incompetence but still contained spectrally detectable haem aa were analysed. The isolated mutant enzymes
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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 I67N is positioned between
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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 a and ligand binding to the binuclear centre was affected. In mutant V380M also the
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midpoint potential of Cu was shifted by 65 mV to the positive. The results indicated for these two mutants changes
B
primarily associated with the binuclear centre, possibly associated with an interference in the routes andror 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. q 1997 Elsevier Science B.V.
Keywords: Yeast; Cytochrome c oxidase; Random mutagenesis; Kinetics; Ligand binding
Abbreviations: EPR, electron paramagnetic resonance; PMSF, phenylmethylsulfonylfluoride; PMS, phenazine methosulphate; DCPIP,
2,6-dichlorophenolindophenol; DAD, 2,3,5,6-tetramethylphenylene diamine; FIRE, flash-induced reduction.
)
Corresponding author. Fax: q49 69 6301 6970; E-mail: brandt@zbc.klinik.uni-frankfurt.de
1
Present address: Department of Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
0005-2728r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.
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PII S0005-2728 97 00035-2