Conformational gating of the electron transfer reaction Q _A ^{− ⋅} Q _B → Q _A Q _B ^{− ⋅} in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

Abstract

The mechanism of the electron transfer reaction, Q A ؊ ⅐ Q B 3 Q A Q B ؊ ⅐ , was studied in isolated reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides by replacing the native Q 10 in the Q A 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 Q A ؊ ⅐ to Q B was measured by three independent methods: a functional assay involving cytochrome c 2 to measure the rate of Q A ؊ ⅐ 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 Q A ؊ ⅐ to Q B 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 Q B by Ϸ5 Å as observed in the x-ray studies of Stowell et al.

Trapped Conformational States of Semiquinone (D+•QB-•) Formed by B-Branch Electron Transfer at Low Temperature in Rhodobacter sphaeroides Reaction Centers

“…The first electron transfer step, which is the focus of this work, occurs with an observed rate constant k AB (1) , which is in the time range 10 4 -10 5 s −1 (9,21,22). The kinetic decay is best described by the sum of several exponential components indicating that the reaction involves more than one step (22,23).…”

“…However, this is unlikely given the independence of the rate on the length of the isoprenoid chain of Q B (9,41,42) and the observation of the same rate constant k AB (1) measured in a mutant RC in which Q B was predominantly structurally located in the proximal site (29). Another possible gating mechanism involves a conformational change in the protein that is required to favor the Q A −• Q B → Q A Q B −• reaction.…”

“…The reaction rate was shown to depend upon protein conformation based on changes of electron transfer upon freezing to cryogenic temperatures (4,5), crosslinking of the protein subunits (6) and embedding the protein in a glassy matrix (7) or PVA film (8). Furthermore, quinone (Q A ) substitution studies showed that the observed rate of electron transfer was independent of the driving force indicating a conformational gating mechanism (9). In this work we investigate the properties of different conformational states of the RC trapped by freezing the protein at cryogenic temperature.…”

“…The kinetic decay is best described by the sum of several exponential components indicating that the reaction involves more than one step (22,23). The independence of the transfer rate on the driving force for electron transfer (9) has led to the proposal that the reaction is conformationally gated (for review see ref. 24).…”

Induced conformational changes upon Cd 2+ binding at photosynthetic reaction centers

“…The PT rate constant belonging to kinetic phase 1 of WT-bRC was estimated to be 10 5 s Ϫ1 (3). It was suggested that the ET corresponding to kinetic phase 1 is coupled to a ''conformational gating'' step governed by protein dynamics, which constitutes the rate-limiting step for k AB (1) (4). The second ET event corresponding to kinetic phase 2 (Eq.…”