Kinetics of the quinone binding reaction at the Q_B site of reaction centers from the purple bacteria Rhodobacter sphaeroides reconstituted in liposomes

Abstract

Transmembrane proton translocation in the photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides is driven by light and performed by two transmembrane complexes; the photosynthetic reaction center and the ubiquinol–cytochrome c oxidoreductase complex, coupled by two mobile electron carriers; the cytochrome and the quinone. This paper focuses on the kinetics and thermodynamics of the interaction between the lipophylic electron carrier ubiquinone‐10 and the photosynthetic enzyme reconstituted in liposomes. The collected data were simulated with an existing recognized kinetic scheme [Shinkarev, V.P. & Wraight, C.A. (1993) In The Photosynthetic Reaction Center (Deisenhofer, J. & Norris, J.R., eds.), pp. 193–255. Academic Press, San Diego, CA, USA] and the kinetic constants of the uptake (7.2 × 107 m−1·s−1) and release (40 s−1) processes of the ligand were inferred. The results obtained for the quinone release kinetic constant are comparable to the rate of the charge recombination reaction from the state D+QA–. Values for the kinetic constants are discussed as part of the overall photocycle, suggesting that its bottleneck may not be the quinone uptake reaction in agreement with a previous report (Gerencser, L., Laczko, G. & Maróti, P. (1999) Biochemistry38, 16866–16875).

Trapping of a long-living charge separated state of photosynthetic reaction centers in proteoliposomes of negatively charged phospholipids

“…This charge-separated state lives 400 times to 10000 times longer than the normally generated D þ Q À B and D þ Q À A states in proteoliposomes (Milano et al 2003) and will be called from now on the LLCS state. When ferrocene (an exogenous electron donor to D + ) was added to freshly prepared PG, PS and PI light-proteolipo- In both panel the peak at 865 nm increases with time following the re-reduction of the bacteriochlorophyll dimer.…”

“…RC reconstitution in PC, PG, PI and PS proteoliposomes was accomplished by size-exclusion chromatography at pH 7 following the procedure outlined in (Kutuzov 1990;Kutuzov et al 1991;Venturoli et al 1991;Trotta et al 2002;Milano et al 2003). This reincorporation method was shown to almost completely remove the detergent bound to the protein (Overfield and Wraight 1980).…”

“…Lipids have been shown to modulate the activities of both bc 1 complex and RC. In particular the charge separated state of RC is largely stabilized in proteoliposomesreconstituted protein (Nagy et al 1998(Nagy et al , 1999Palazzo et al 2000;Trotta et al 2002;Milano et al 2003) with respect to detergent solubilized protein (Kleinfeld et al 1984). Similarly the enzymatic activity of detergent solubilized cytochrome bc 1 complex is greatly enhanced (Andrews 1990) in presence of phosphatidylcholine.…”

Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres*

“…This can be done through sets of kinetic equations modeling the charge transfer kinetics within the RC protein and the binding/unbinding equilibrium dynamics of the quinone and inhibitor molecules. Indeed, although similar investigations have already been carried out so far, with reasonable results, these provide only limited theoretical interpretation to a restricted set of experimental results, without particular attention to the presence of herbicide inhibitors (Halmschlager et al 2002;Milano et al 2003). Nevertheless, this information may be significant for several applications.…”

“…While UQ-10 on-rate (k Q,on ) and off-rate (k Q,off ) constants were estimated as 7 × 10 7 M -1 ·s -1 and 40 1 s -1 , respectively (Milano et al 2003), the same parameters for terbutryn were (1÷8) × 10 4 M -1 ·s -1 and about 0.02 s -1 , with a difference of three orders of magnitude (Baker and Percival 1991). Future developments of these studies will be oriented also towards the construction of a suitable kinetic simulation model of the RC-quinoneherbicide dynamics from experimental data, in order to obtain reliable on-rate and off-rate parameters (see Future Perspectives section).…”