Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase:  Is the Reaction Electroneutral?

Abstract

Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O 2 . Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a 3 and Cu B . If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK a s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu B shifts its pK a from below 7 to above 7 and so picks up a proton when heme a 3 and Cu B are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a 3 all have pK a s above 7 in all redox states, although they have only 1.6-3.5 ∆pK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK a s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu A , heme a, heme a 3 , and Cu B shows ≈2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E m ) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E m of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E m is independent of the BNC redox state.Heme-copper oxidases are the terminal electron acceptors in anaerobic organisms. These transmembrane proteins reduce dioxygen and convert the released chemical energy into an electrochemical gradient, across the eukaryotic mitochondrial membrane or the bacterial cell membrane (1-4). Cytochrome c oxidases are the most prevalent hemecopper oxidases. In this protein 4 electrons, provided by 4 cytochromes c, are used to reduce dioxygen to water. The 4 protons needed to make water are taken from the negative cytoplasmic side of the membrane, adding to the electrochemical gradient. Four additional protons are pumped across the membrane (5). Thus, each dioxygen molecule reduced by cytochrome c oxidase is coupled to the transfer of 8 charges across the membrane.The first step of electron transfer is from cytochrome c to Cu A , a dicopper center in subunit II which extends beyond the membrane on the proton release side of the protein (Figure 1). After receiving the electron, Cu A reduces the 6-coordinate, low-spin, bis-Hi...

Electrostatic Environment of Hemes in Proteins: pKas of Hydroxyl Ligands

“…This raises all ferric heme pK a s by 0.74 pH unit and raises the aquo-heme E m s by 30 mV. Systematic shifts are also applied to the ionized form of ionizable amino acids on the basis of pK a benchmark studies (43,56,65).…”

“…including the change in reaction field energy from solution (∆∆G rxn ), the added interaction with the rigid backbone (∆G pol ), and the side chains in their ionization and conformation states equilibrated at the given pH and E h (∆G res ) (20,43,77).…”

“…The influence of the charge distribution of Cu B is more complex and cannot be described by a simple scaling factor. The details are discussed in ref 43.…”

“…The scaling factor derived for particular heme-residue geometry from the comparison of Coulomb's law and DFT interactions in a vacuum (Figure 2) is used to scale the individual PB interactions in MCCE. Corrections used for Cu B can be found in ref 43.…”

“…The aquo-heme E m is not a factor in the function of myoglobin or hemoglobin, nor are the pK a s important for the reaction mechanism of most of the proteins studied here. However, the aquoheme pK a in cytochrome c oxidase appears to determine whether heme a 3 binds a proton during the reduction of the binuclear center (43). Thus, this pK a helps to control the electroneutrality of the oxidase reduction.…”