Analysis of site-directed mutants locates a non-redox-active metal near the active site of cytochrome c oxidase of Rhodobacter sphaeroides.
Substoichiometric amounts of Mn are bound by the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and appear in the EPR spectrum of the purified enzyme as signals that overlay those of CuA in the g = 2.0 region. The Mn is tightly bound and not removed by a high degree of purification or by washing with 50 mM EDTA. The amount of bound Mn varies with the ratio of Mg to Mn in the growth medium. Oxidase containing no EPR-detectable Mn can be prepared from cells grown in low Mn/Mg, while high Mn/Mg in the growth medium gives rise to near stoichiometric levels (0.7 mol/mol of aa3). Incubation of purified Mn-deficient oxidase with 1 mM Mn does not allow incorporation into the tight binding site, indicating that this site is not accessible in the assembled protein. When bound Mn is depleted by growth in high Mg, there is no change in electron transfer activity, suggesting that Mg may substituted for Mn and maintain protein structure. Analysis of site-directed mutants in an extramembrane loop close to the active site of cytochrome oxidase identifies His-411 and Asp-412 of subunit I as probable ligands of the Mn. Mutation of either residue leads to lower activity and loss of Mn binding, even in cells grown in elevated concentrations of Mn. Since Mn binding correlates with the [Mn] to [Mg] ratio in the culture medium, we propose that Mn competes for the site that normally binds a stoichiometric Mg ion in aa3-type cytochrome c oxidases.
Hosler
J P
JP
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Espe
M P
MP
Zhen
Y
Y
Babcock
G T
GT
Ferguson-Miller
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D008667:Metalloproteins; D008345:Manganese; D006639:Histidine; D003576:Electron Transport Complex IV; D008274:Magnesium