Protein Tuning of Ligand Pathways and Metal Centers in Heme-Copper Oxidases

Abstract

Knowledge of how the protein environment can tune ligand pathways and metal-based prosthetic group(s) is fundamental for understanding the catalytic functions of metallo-enzymes. The structural and functional similarities and differences among the heme-copper oxidases, which play a key role in energy production of aerobic organisms, make them an ideal system for exploring the effect of the protein environment on enzymatic function. For example, the catalytic subunit of the Rhodobacter sphaeroides (Rs) and Paracoccus denitrificans (Pd) aa3 oxidases has high sequence homology to its mitochondrial counterpart, while that of other oxidases, including Thermus thermophilus (Tt) ba3, is much lower. To establish possible relationships between the structural diversity of these enzymes and their ligand binding dynamics, we used photolabile O2 and NO complexes to monitor the kinetics of O2 and NO reacting with reduced wild-type Thermus thermophilus (Tt) ba3 and the bovine heart aa3 oxidase. Time-resolved optical absorption measurements show that O2 and NO bind to reduced heme a3 in Ttba3 with a second-order rate constant of 1x109 M−1s−1. This rate is 10-times faster than observed for the mammalian enzyme, suggesting that inherent structural differences affect ligand access in the two enzymes. To test this hypothesis, we used x-ray crystallography, time-resolved optical absorption spectroscopy, and theoretical calculations to study ligand access in Tt ba3 mutants, in which tyrosine and/or threonine in the O2-channel of Tt ba3 were replaced by the corresponding bulkier tryptophan and phenylalanine residues of the aa3 enzymes. The results are consistent with a constriction point and hydrophobic pocket in the O2-channel of the bovine aa3 impeding access of NO and O2 to the active site in this enzyme but not in Tt ba3. Such structural differences may reflect evolutionary adaptation of the two enzymes to different environments.