Probing the Q-Proton Pathway of ba3-Cytochrome c Oxidase by Time-Resolved Fourier Transform Infrared Spectroscopy

Abstract

In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. Two proton pathways (K and D) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases. The Q pathway starts from the cytoplasmic side of the membrane and leads through the axial heme a3 ligand His-384 to the propionate of the heme a3 pyrrol ring A, and then via Asn-366 and Asp-372 to the water pool. We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopies to investigate the protonation/deprotonation events in the Q-proton pathway at ambient temperature. The photolysis of CO from heme a3 and its transient binding to CuB is dynamically linked to structural changes that can be tentatively attributed to ring A propionate of heme a3 (1695/1708 cm(-1)) and to deprotonation of Asp-372 (1726 cm(-1)). The implications of these results with respect to the role of the ring A propionate of heme a3-Asp372-H2O site as a proton carrier to the exit/output proton channel (H2O pool) that is conserved among all structurally known heme-copper oxidases, and is part of the Q-proton pathway in ba3-cytochrome c oxidase, are discussed.

Mutation of a single residue in the ba 3 oxidase specifically impairs protonation of the pump site

“…Results from earlier studies using infrared spectroscopy indicated a link between ligand binding and changes in structure and/or protonation around Asp372 (31,32). The CO-photolysis data in Fig.…”

“…Fourier transform infrared (FTIR) data from earlier studies of the ba 3 CytcO indicated that ligand binding to heme a 3 is linked to structural or protonation changes around Asp372 (31,32). Therefore, here we compared the kinetics of CO recombination in the reduced wildtype and Asp372Ile variant of ba 3 CytcO from T. thermophilus (Fig.…”

“…Earlier FTIR spectroscopy data suggested that a possible acceptor for pumped protons is a cluster involving Asp372, a water molecule, and the ring A propionate of heme a 3 (31). The same protein segment was suggested on the basis of theoretical calculations, which indicated that His376, which is also hydrogenbonded to the ring A propionate, may accept protons from the n side of the membrane (29).…”

“…1). For the ba 3 CytcO, theoretical and experimental data, together with structural analyses, suggest that the ring A propionate of heme a 3 , including nearby sites, may act as the PLS (12,15,27,29,31,32). Results from a more recent study showed that structural perturbations near the heme a 3 propionate A resulted in uncoupling of proton pumping from O 2 reduction.…”

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3fromThermus thermophilus

“…Varotsis and coworkers (35) postulated a critical role in proton pumping of cytochrome ba 3 from T. thermophilus for the hydrogen-bonded carboxylate pair of propionate-A of heme a 3 and D372 (Fig. 1).…”