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

Abstract

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617–11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

All the O2 Consumed by Thermus thermophilus Cytochrome ba3 Is Delivered to the Active Site through a Long, Open Hydrophobic Tunnel with Entrances within the Lipid Bilayer

“…In contrast, the inferred pathway from X-ray data for O 2 diffusion to the active site of cytochrome ba 3 is supported by both previous experimental data and by MD simulations, which only concentrated on the dynamics and stability of O 2 binding at individual X-ray Xe sites, 33,34 as well as the present study. The tunnel in this B-family heme-copper oxygen reductase does not have the constriction observed in the A-family enzymes.…”

“…Short (1 ns) MD simulations showed that there is effectively no barrier within this tunnel for O 2 diffusion. 34 …”

“…The results of both the ELS and ILS calculations are in full agreement with each other as well as previous experimental and computational studies. 33,34 The most important points are summarized below.…”

“…6,7,22–25 In contrast to the electron and protons “wires”, pathways for O 2 diffusion into the active site have not been well-characterized. Hydrophobic tunnels in the X-ray structures of the aerobic respiratory heme-copper oxygen reductases, 26–32 including cytochrome ba 3 from Thermus thermophilus , 28,31,33,34 have been suggested as putative O 2 tunnels. Xenon (Xe), which has a similar size as O 2 , is often used in crystallographic studies to locate hydrophobic cavities that may provide binding sites for small diatomic gas molecules, including O 2 , CO, H 2 or N 2 .…”

Glyphosate pathways to modern diseases V: Amino acid analogue of glycine in diverse proteins

“…Subunit II of COX contains a Cu A redox centre, serves as a binding partner for cytochrome c, and as a participant in the electron transfer process [86]. Subunit II has a highly conserved glycine residue at the active site [87][88][89]. A mutant form of COX in Rhodobacter sphaeroides involving a substitution of valine for the conserved gly283 resulted in a complete block of access of oxygen to the active site [88].…”