Conformational gating of the electron transfer reaction QA-.QB --> QAQB-. in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

Abstract

The mechanism of the electron transfer reaction, QA-.QB --> QAQB-., was studied in isolated reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides by replacing the native Q10 in the QA binding site with quinones having different redox potentials. These substitutions are expected to change the intrinsic electron transfer rate by changing the redox free energy (i.e., driving force) for electron transfer without affecting other events that may be associated with the electron transfer (e.g., protein dynamics or protonation). The electron transfer from QA-. to QB was measured by three independent methods: a functional assay involving cytochrome c2 to measure the rate of QA-. oxidation, optical kinetic spectroscopy to measure changes in semiquinone absorption, and kinetic near-IR spectroscopy to measure electrochromic shifts that occur in response to electron transfer. The results show that the rate of the observed electron transfer from QA-. to QB does not change as the redox free energy for electron transfer is varied over a range of 150 meV. The strong temperature dependence of the observed rate rules out the possibility that the reaction is activationless. We conclude, therefore, that the independence of the observed rate on the driving force for electron transfer is due to conformational gating, that is, the rate limiting step is a conformational change required before electron transfer. This change is proposed to be the movement, controlled kinetically either by protein dynamics or intermolecular interactions, of QB by approximately 5 A as observed in the x-ray studies of Stowell et al. [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E. & Feher, G. (1997) Science 276, 812-816].

Figure 1

Optical absorbance changes at 412 nm associated with electron transfer from Q_A^−⋅ to Q_B in hybrid (Me₃NQ^−⋅ or MQ_P^−⋅ in the Q_A site and Q₁₀ in the Q_B site) and native (Q₁₀ in both Q_A and Q_B sites) RCs. In native RCs, the absorbance change is due to an electrochromic shift of the BPhe absorption. In hybrid RCs, the main effect is caused by the difference in extinction coefficient between the naphthosemiquinones (NQ^−⋅ in the Q_A site and Q₁₀^−⋅ in the Q_B site). The observed transients in hybrid RCs are reduced by the electrochromic shift, which was assumed to be the same as that observed in native RCs. The data for the hybrid RCs were normalized to the expected absorption determined from the known difference in extinction coefficients between (NQ)_A^−⋅ and Q₁₀^−⋅ (11, 20, 21). A shallow slope determined by monitoring the recombination reactions (D⁺Q_A^−⋅ → DQ_A and D⁺Q_AQ_B^−⋅ → DQ_AQ_B) was subtracted for clarity. Addition of terbutryne, which inhibits electron transfer to Q_B, eliminated the transient kinetic phases. [Conditions: 5 μM 2.4.1RCs, BMK buffer (pH = 7.2), 0.03% LDAO. Spectral bandwidth, 10 nm].

Figure 2

Optical absorbance changes at 865 nm for native RCs in the presence of cyt c₂²⁺ after two laser flashes spaced Δt apart. Laser flash artifacts are truncated. The ratio of ΔA₂ to ΔA_{No cyt} gives the fraction of photoactive RCs at the time of the second flash (see Eq. 3). ΔA₁ is due to the fraction of RCs that do not have a bound cyt c₂²⁺. Addition of terbutryne to inhibit electron transfer to Q_B eliminated the absorbance change in response to the second laser flash. (Conditions: 3 μM 2.4.1 RCs with Q₁₀ reconstituted into the Q_A and Q_B sites by addition of Q₁₀ in LDAO. After reconstitution, LDAO was 0.03%. 100 μM cyt c₂²⁺, 5 μM ascorbate, 1 mM Tris, and 0.04% maltoside; spectral bandwidth, 10 nm.)

Figure 3

The fraction of photoactive RCs as a function of the time interval between two laser flashes for native (•), and hybrid (□) RCs. The solid line represents a least squares biexponential fit to the data. A minimum of 20 min dark recovery was allowed between sets of flashes. (865 nm assay conditions given in Fig. 2; at 550 nm, cyt c₂²⁺ concentration was reduced to ≈30 μM; spectral bandwidth, 4 nm.)

Figure 4

The electron transfer rate k_AB⁽¹⁾ as a function of redox free energy (driving force) for electron transfer (data from the second and seventh columns of Table 1). The solid line gives a best fit to the data. The dashed line represents the dependence predicted by the Marcus theory for an electron transfer limited reaction using the measured free energy difference between the initial, Q_A^−⋅Q_B, and final, Q_AQ_B^−⋅, states of 60 meV (19) and 1.1 eV for the reorganization energy (13, 31). Error bars represent one SD of the mean. Quinones incorporated into the Q_A site listed in order of decreasing redox potential were: 1, MQ₀; 2, Q₁₀; 3, MQ_P; 4, MQ₄; 5, Me₃NQ; and 6, Me₄NQ. Redox energies are quoted with respect to Q₁₀ (see • without error bars).

Figure 5

Proposed model for conformational gating of electron transfer from Q_A^−⋅ to Q_B. Two conformational states are shown, distal (D; oval), which is inactive, and proximal (P; rectangle), which is active in electron transfer. The main sequence of steps in the reaction are (1) excitation of RCs in the distal state, (2) conversion to the proximal state, and (3) electron transfer. A fraction of RCs in the proximal state before light excitation may give rise to a faster observed rate through reactions 1′ and 3.

Figure 6

Comparison of the binding positions for Q_B determined from the light (D⁺Q_A Q_B^−⋅) and dark (DQ_AQ_B) x-ray crystal structures of the RC (10). Movement from an inactive–distal (black) to an active–proximal (gray) binding site is proposed as the major structural change involved with conformational gating of electron transfer from Q_A^−⋅ to Q_B. Hydrogen bonding partners are connected by dotted lines. Modified from ref. .