Conserved Glycine 232 in the Ligand Channel of ba3 Cytochrome Oxidase from Thermus thermophilus

Heme-copper oxidases play a key role in mitochondrial and bacterial respiration, catalyzing the reduction of dioxygen to water and redox-coupled proton translocation.1−3 The resulting electrochemical gradient is used by ATP synthase to make ATP.4 The heme-copper oxidases are classified into the A, B, and C subfamilies.5,6 Ligand channels have been proposed for oxidases from the different subfamilies, including for the bovine enzyme,7Rhodobacter sphaeroides (Rs) aa3,8Paracoccus denitrificans (Pd) aa3,9 and Escherichia coli bo3(10) from the A family, Thermus thermophilus (Tt) ba3 from the B family,11 and Pseudomonas stutzeri cbb3 from the C family.12 A hydrophobic channel originating at a V-shaped cleft formed by subunit III was identified in the crystal structure of Pd aa3.9 A channel starting in subunit I and converging with the channel starting in subunit III was observed in Rs aa3 by trapping Xe atoms in the crystal structure.8 More recently, additional details of the ligand channel in Tt ba3 have been obtained.13,14 The location of Xe atoms in the Tt ba3 crystal structure shows a bifurcated Y-shaped ligand channel 18–20 A in length, starting from two points, the first between helices II and III of subunit I and the second between helices IV and V of subunit I on the protein exterior.13,14 On the basis of these observations, cavities in the bovine, Rs, and Pd aa3 oxidases were assessed and putative O2 pathways suggested.13 In the bovine, Rs, and Pd aa3 oxidases, there is a narrowing of the ligand channel ∼9 A from the catalytic site, caused by conserved tryptophan and phenylalanine residues;8,15,16 this narrowing is not present in Tt ba3 in which smaller residues, tyrosine (Y133) and threonine (T231), occupy the positions of the tryptophan and phenylalanine, respectively.11,13 Recent experiments in our laboratory have shown that binding of O2 and NO to the active site in the Y133W and Y133W/T231F mutants of Tt ba3 is ∼5 times slower than in the wild-type enzyme.17 This suggests that the significantly slower ligand binding in the bovine enzyme (1 × 108 M–1 s–1) compared to that in Tt ba3 (1 × 109 M–1 s–1)18,19 is in part due to the tryptophan constriction residue in the ligand channel of the bovine aa3 enzyme (W126) impeding the access of O2 and NO to the active site.17 A hydrophobic pocket in the ligand channel of the bovine enzyme may further impede the access of O2 and NO to the active site.17 A comparison of amino acid sequences and crystal structures of the heme-copper oxidases has also identified strictly conserved residues in the ligand channels of these enzymes, including that of Tt ba3, which may play important roles in modulating access of ligands to the binuclear active site. One of the highly conserved amino acid residues in the ligand channel is a glycine residue, G232 in Tt ba3.8,10,11,20,21 The crystal structure of Tt ba3 shows that G232 is located between two large residues, tryptophan (W229) and phenylalanine (F228) (Figure ​(Figure1), which1), which have been suggested to form a hydrophobic and hydrophilic boundary around the binuclear center.11,13 The G232 residue is located behind the Xe1 site, identified in Xe-pressurized crystallographic studies of Tt ba3,13,14 providing a small opening proposed to be the site of entry of ligands into the active site. Mutation of the homologous glycine residue in Rs aa3 oxidase (G283) to a larger valine residue was reported to give rise to significantly faster CO recombination [major phase (50%), 40 μs, compared to 10 ms in the wild-type enzyme], suggesting that the photodissociated CO did not leave the catalytic site cavity of the mutant enzyme.16 The oxidation of the fully reduced G283V mutant upon exposure to dioxygen was found to be significantly slower (minutes) compared to that of the wild-type enzyme (1 ms), which was interpreted as the valine side chain completely blocking access of O2 to the catalytic site in this enzyme.16 Because of the critical involvement of this conserved residue in ligand access in Rs aa3, it is important to know if G232 plays a similar role in Tt ba3, particularly considering that the G232 residue is adjacent to T231 Tt ba3, which in the aa3 oxidases is replaced by the phenylalanine “constriction point” residue. Figure 1 Residues lining the ligand channel of the wild-type ba3 cytochrome oxidase (colored yellow, PDB entry 3S8F) and the G232V mutant (blue). The purple spheres are water 618 and the green spheres water 604 as described in the text. In this study, the CO photolysis and recombination dynamics and the reactions of O2 and NO with the G232V Tt ba3 mutant were investigated using photolabile NO and O2 carriers. The replacement of glycine with the larger valine does not impede ligand access because the resulting structural change appears to open other O2 cavities in the ligand channel.