Rhodobacter capsulatus?ta bulunan cbb3 oksidazın I. alt ünitesinin IX. heliksi üzerindeki oldukça korunmuş rezidülerinin yönlendirilmiş mutagenezi

Oxygen reductases are terminal respiratory oxidases in mitochondria and many aerobic bacteria and archaea. Site-directed mutagenesis experiments and the crystal structures of the aa3-type oxygen reductases revealed two proton transfer pathways, named the D- and the K-pathways. Aspartic acid 361 (D361), glutamate 380 (E380) and lysine 387 (K387) are highly conserved amino acid residues (R. capsulatus numbering) in the subunit I of the aa3 and ba3?type oxygen reductases. Mutations at glutamate 380 (E380) locus have been shown to block the oxygen reduction and the uptake of protons, suggesting that this residue plays an important role in the catalytic mechanism of aa3 type oxidases. D361 and K387 are also thought that highly conserved amino acids especially in aa3 type oxidase and play important roles on enzyme activity.Moreover, thermodynamic and spectroscopic properties of the redox centers in oxygen reductases indicated that these three residues were important for histidine-glutamate/aspartate structural motifs found in a variety of enzymes. A sequence comparison of the catalytic subunits shows that these residues are not conserved within VIII. helix of cbb3-type oxygen reductase. However, homology modeling has suggested that D361, E380 and K387 are located within helix IX of subunit I and fully conserved among the cbb3-type oxygen reductases, might fulfill this function.In the present study, in order to understand molecular mechanism of proton pumping channel, conserved aspartic acid, glutamic acid and lysine residues within helix IX of subunit I of the cbb3-type oxygen reductases from R. capsulatus were substituted for asparagine 361 (N361), glutamine 380 (Q380) and methionine 387 (K387) respectively by site directed mutagenesis. The effects of these mutations on enzyme activity were evaluated by NADI staining and it was found that while the E380Q mutant was completely inactive, other D361N and K387M mutant had wild type activity. The result supports the proposal that the cbb3-type oxygen reductases have a proton-conducting channel that is analogous to the D-channel in the aa3-type oxygen reductases.