The Redox-Active Tyrosine Is Essential for Proton Pumping in Cytochrome c Oxidase

Abstract

Cellular respiration involves electron transport via a number of enzyme complexes to the terminal Cytochrome c oxidase (CcO), in which molecular oxygen is reduced to water. The free energy released in the reduction process is used to establish a transmembrane electrochemical gradient, via two processes, both corresponding to charge transport across the membrane in which the enzymes are embedded. First, the reduction chemistry occurring in the active site of CcO is electrogenic, which means that the electrons and protons are delivered from opposite sides of the membrane. Second, the exergonic chemistry is coupled to translocation of protons across the entire membrane, referred to as proton pumping. In the largest subfamily of the CcO enzymes, the A-family, one proton is pumped for every electron needed for the chemistry, making the energy conservation particularly efficient. In the present study, hybrid density functional calculations are performed on a model of the A-family CcOs. The calculations show that the redox-active tyrosine, conserved in all types of CcOs, plays an essential role for the energy conservation. Based on the calculations a reaction mechanism is suggested involving a tyrosyl radical (possibly mixed with tyrosinate character) in all reduction steps. The result is that the free energy released in each reduction step is large enough to allow proton pumping in all reduction steps without prohibitively high barriers when the gradient is present. Furthermore, the unprotonated tyrosine provides a mechanism for coupling the uptake of two protons per electron in every reduction step, i.e. for a secure proton pumping.

Keywords: cytochrome c oxidase; density functional theory; energy conservation; midpoint potentials; proton pumping; redox-active tyrosine.

FIGURE 1

Overview of the A-family CcOs. The BNC active site cofactors: high-spin heme a ₃, Cu_B and the cross-linked tyrosine are embedded in the inner mitochondrial or the bacterial membrane. Soluble cytochrome c, the electron transfer cofactors (Cu_A and low-spin heme a), plus proton channels (D and K) from the N-side of the membrane to the BNC are indicated. Electron and proton uptake to the BNC is indicated as arrows, as well as the proton pumping across the entire membrane.

FIGURE 2

Model of the active site in CcO used in the present calculations, showing the optimized F state.

FIGURE 3

One reduction step, scetching the main steps in a commonly accepted scheme for proton pumping in CcO (Wikström et al., 2018). 1. Electron transfer from soluble cytochrome c to the low-spin heme a. The EA of the BNC is too low for the electron to move further into the BNC. 2. The electron in heme a triggers proton uptake from the N-side to PLS, which increases the EA of the BNC, such that the electron can move from heme a into the BNC. 3. The electron in the BNC triggers the uptake of the chemical proton from the N-side to the BNC, and the proton in the BNC repels the proton in the PLS, which is ejected to the P-side. - The F to O _H reduction is used as an example.

FIGURE 4

Calculated electron affinities (EA, kcal/mol) of possible intermediates in the catalytic cycle of the A-family CcOs. (A) The mechanism for O₂ reduction suggested in Table 2 with calculated EAs for the intermediates that initiate each of the reduction steps. (B) Calculated EAs for alternative structures for each reduction step.

FIGURE 5

Scetch of energy profiles for coupled electron transfer from heme a to the BNC and proton transfer from the N-side to PLS (full curves). The dotted lines correspond to only electron transfer from heme a to the BNC. (A) For the F state, with a high EA, the coupled reaction is exergonic. (B) For the F′ state, with a low EA, the coupled reaction is endergonic.