Modification of quinone electrochemistry by the proteins in the biological electron transfer chains: examples from photosynthetic reaction centers

Abstract

Quinones such as ubiquinone are the lipid soluble electron and proton carriers in the membranes of mitochondria, chloroplasts and oxygenic bacteria. Quinones undergo controlled redox reactions bound to specific sites in integral membrane proteins such as the cytochrome bc(1) oxidoreductase. The quinone reactions in bacterial photosynthesis are amongst the best characterized, presenting a model to understand how proteins modulate cofactor chemistry. The free energy of ubiquinone redox reactions in aqueous solution and in the Q(A) and Q(B) sites of the bacterial photosynthetic reaction centers (RCs) are compared. In the primary Q(A) site ubiquinone is reduced only to the anionic semiquinone (Q(*-)) while in the secondary Q(B) site the product is the doubly reduced, doubly protonated quinol (QH(2)). The ways in which the protein modifies the relative energy of each reduced and protonated intermediate are described. For example, the protein stabilizes Q(*-) while destabilizing Q(=) relative to aqueous solution through electrostatic interactions. In addition, kinetic and thermodynamic mechanisms for stabilizing the intermediate semiquinones are compared. Evidence for the protein sequestering anionic compounds by slowing both on and off rates as well as by binding the anion more tightly is reviewed.

Fig. 1

The E_ms on the horizontal arrows are in millivolt. The pK_as, are in parenthesis on the vertical arrows. The quinone species in the dark circles are found in both the Q_A and Q_B sites. Thin circles indicate species found only in the Q_B site. The species in grey are not seen in RCs or in aqueous solution as they are expected to have pK_as below pH 0. All values are taken from (Zhu and Gunner 2005, see references therein)

Fig. 2

Modification of quinone E_ms by proton or protein binding. a Energetics of coupled electron and proton transfer. X is a base associated with a quinone binding site. When X is neutral the E_m for Q is E_m,0; when Q is neutral the pK_a for X is pK_a,0. Favorable interactions between X⁺ and Q^•− raise the E_m for Q by −ΔΔG°/nF. The same interaction stabilizes XH⁺, raising the pK_a by ΔΔG°/2.3RT. A ΔΔG° of −1.36 kcal/mol shifts the E_m up by 58 mV and the pK_a up by 1 pH unit. If the pK_a of X remains below the pH in the presence of Q^•− then X will never be protonated and E_m=E_m,0. If the pH is below the pK_a of X with Q oxidized, XH⁺ will be present throughout the reaction and the observed E_m will be E_m=E_m,0−ΔΔG°/F. In this case the reduction of Q feels the full stabilization by the adjacent base. In either case the reaction is pH independent. However, if the pH is at least ≈2 pH units below the pK_a with Q and ≈2 pH units above the pK_a in the presence of Q^•− (i.e. ΔΔG° >≈5.4 kcal/mol) then binding ≈1 proton will be coupled to electron transfer (when the pH is 2 pH units below the pK_a X will remain 1% protonated; when the pH is 2 pH units above the pK_a X will remain 1% XH⁺. Thus, on average 0.98 more protons will be bound). The resultant E_m will be E_m,0− [ΔΔG°+(pH−pK_a,0)/2.3RT]/F. The free energy needed to protonate X at this pH is (pH−pK_a,0)/2.3RT. It is this term which leads to the classic 60 meV/pH unit E_m shift with pH indicative of 1 proton bound/electron. The cost of rearranging the surroundings diminishes the E_m shift from that found if XH⁺ were present at the start of the reaction. If pK_a,0 for X is near the pH even a small ΔΔG° of interaction with a more distant Q^•− will lead to substoichiometric changes in protonation of X, leading to a pH dependence smaller then 60 mV/pH unit. b The relationship between the thermodynamics of quinone binding and quinone electrochemistry. If the E_m is more positive in the quinone binding site then the semiquinone is bound more tightly then the quinone. ΔΔG°_bind is 2.3RT(logK_dQ^•−− logK_dQ)

Fig. 3

The overview of the sequence of electron transfer reactions in the bacterial type II reaction centers. The reaction starts with a dimer of bacteriochlorophylls (P) excited by light. Electron transfer from the excited state (P*) leads to reduction of a nearby bacteriopheophytin (H). The reduced H^•− is used to reduce the primary quinone, Q_A, which in turn reduces the secondary quinone, Q_B, to the anionic semiquinone (Q_B^•−). Absorption of a second photon again leads to formation of Q_A^•− which now reduces Q_B^•− to the quinol, QH₂, which dissociates from the protein and quinone is rebound. The Q_B charge in parenthesis is found on the second turnover. The order in which electrons and protons are added to Q_B is described in Fig. 4 B. In the membrane of the purple non-sulfur photosynthetic bacteria the cytochrome c is rereduced and the QH₂ reoxidized by the cytochrome bc₁ oxidoreductase with the concomitant increase in the transmembrane proton gradient

Fig. 4

Energy levels for sequential electron transfer from a primary semiquinone to a secondary quinone. A Q_AS:Q_BS indicate a complex of two quinones which have the same electrochemistry as two isolated ubiquinones in solution at pH 7 (Fig. 1). To start the cycle one quinone is reduced to the semiquinone forming Q_AS^•−:Q_BS. Given a solution E_h of 0 mV or a chemical electron donor with an E_m of 0 mV, the single reduction of quinone with an E_m of −145 mV to Q^•− is uphill by 145 meV. Since these quinones are identical, electron transfer from Q_AS^•− to Q_BS will be isoenergetic. Given the semiquinone pK_a of 4.9, protonating either semiquinone would be uphill by ≈120 meV. Thus, the lowest energy, singly reduced state will be a 50:50 mixture of Q_AS^•−:Q_BS and Q_AS:Q_BS^•−. The second turnover starts with the second reduction of Q_AS forming Q_AS^•−:Q_BS^•−. The thermodynamically preferred pathway has the electron transfer occurring before the first proton is bound. The formation of Q_AS:Q_BS⁼ requires only 55 meV because the second reduction of Q_BS is coupled to the favorable oxidation of Q_AS. The reaction path where Q_BS^•− is protonated to form Q_AS^•−:Q_BSH before it is reduced is 120 meV uphill. Q_AS:Q_BSH⁻ where one quinone has two electrons and one proton is 290 meV lower in energy then Q_AS^•−:Q_BS^•− and is essentially isoenergetic with the initial Q_AS:Q_BS state at pH 7. Once Q_AS:Q_BSH⁻ is formed the second protonation to form Q_AS:Q_BSH₂ is downhill by −220 meV. B The ubiquinone energy levels in R. sphaeroides RCs at pH 7 and E_h 0. In the initial reaction Q_A is reduced to the semiquinone forming Q_A^•−:Q_B. Calculations put this state near 0 mV, close to the measured values between −45 and −70 mV (all calculated values are from Zhu and Gunner (2005)). Q_B^•− is stabilized by 30 (calculation) to ≈70 meV more then Q_A^•−. The second turnover starts with formation of Q_A^•−:Q_B^•−. Calculations and the experiments of Graige and Okamura show that in the protein Q_A^•−:Q_B^•H is lower in energy then Q_A:Q_B⁼ (Graige et al. 1998). The calculations place the energy of Q_A^•−:Q_B^•H 260 meV above Q_A^•−:Q_B^•−, while the kinetics of forward electron transfer support a value of 160 meV (grey text; Graige et al. 1999). The second reduction of Q_BH⁻ is favorable. The anionic Q_AQ_BH⁻ is 110 meV more stable in the protein then in solution, while Q_AQ_BH₂ is 50 meV less stable favoring quinol dissociation