Weak temperature dependence of P (+) H A (-) recombination in mutant Rhodobacter sphaeroides reaction centers

International audienceIn contrast with findings on the wild-type Rhodobacter sphaeroides reaction center, biexponential P (+) H A (-) → PH A charge recombination is shown to be weakly dependent on temperature between 78 and 298 K in three variants with single amino acids exchanged in the vicinity of primary electron acceptors. These mutated reaction centers have diverse overall kinetics of charge recombination, spanning an average lifetime from ~2 to ~20 ns. Despite these differences a protein relaxation model applied previously to wild-type reaction centers was successfully used to relate the observed kinetics to the temporal evolution of the free energy level of the state P (+) H A (-) relative to P (+) B A (-) . We conclude that the observed variety in the kinetics of charge recombination, together with their weak temperature dependence, is caused by a combination of factors that are each affected to a different extent by the point mutations in a particular mutant complex. These are as follows: (1) the initial free energy gap between the states P (+) B A (-) and P (+) H A (-) , (2) the intrinsic rate of P (+) B A (-) → PB A charge recombination, and (3) the rate of protein relaxation in response to the appearance of the charge separated states. In the case of a mutant which displays rapid P (+) H A (-) recombination (ELL), most of this recombination occurs in an unrelaxed protein in which P (+) B A (-) and P (+) H A (-) are almost isoenergetic. In contrast, in a mutant in which P (+) H A (-) recombination is relatively slow (GML), most of the recombination occurs in a relaxed protein in which P (+) H A (-) is much lower in energy than P (+) H A (-) . The weak temperature dependence in the ELL reaction center and a YLH mutant was modeled in two ways: (1) by assuming that the initial P (+) B A (-) and P (+) H A (-) states in an unrelaxed protein are isoenergetic, whereas the final free energy gap between these states following the protein relaxation is large (~250 meV or more), independent of temperature and (2) by assuming that the initial and final free energy gaps between P (+) B A (-) and P (+) H A (-) are moderate and temperature dependent. In the case of the GML mutant, it was concluded that the free energy gap between P (+) B A (-) and P (+) H A (-) is large at all times

Selected electron transfer reactions in reaction centers of purple bacteria studied by transient absorption spectroscopy

Wydział FizykiW pracy przedstawiono wyniki badania początkowych reakcji przeniesienia elektronu w centrach reakcji (CR) bakterii purpurowych: otwartych, charakteryzujących się swobodnym przepływem elektronów, i zamkniętych, w których naturalna ścieżka transportu elektronu została zablokowana na jednym z etapów. Przebadano CR z dzikiego szczepu oraz z zestawu szczepów zmutowanych tak, aby zmienić wartość przerwy energetycznej pomiędzy P+BA- a P+HA-, gdzie P jest donorem, a BA i HA są kolejnymi akceptorami elektronu. Celem pracy było skonstruowanie modelu rekombinacji ładunku spójnego z otrzymanymi wynikami eksperymentalnymi. Praca składa się z dwóch głównych części: wprowadzenia, zawierającego podst. informacje o fotosyntezie, bakteriach purpurowych, CR, a także o przebadanych próbkach i metodach pomiarowych. Część druga prezentuje wyniki i wnioski z wykonanych pomiarów, z których za najważniejsze uznać można obserwację stanu P+BA- w niezmutowanych CR oraz kilkuwykładniczy zanik stanu P+HA- w zamkniętych CR, związany z rekombinacją ładunków. Na podstawie przedstawionych wyników oszacowano wartość przerwy energetycznej ΔG między stanami P+BA- i P+HA- zarówno w skali czasu rozdziału ładunku (kilka ps), jak i rekombinacji (kilkaset ps – kilkanaście ns), oraz skonstruowano spójny model rekombinacji ładunku w zamkniętych CR, zgodnie z którym rekombinacja zachodzi za pośrednictwem stanu P+BA-, a poziom stanu P+HA- względem P+BA- obniża się w czasie w wyniku relaksacji – biernej odpowiedzi białka na pojawiające się w układzie ładunki. Relaksacja i rekombinacja są procesami kompetycyjnymi, zachodzącymi w tej samej skali czasu.This dissertation shows results of studies of primary charge transfer in reaction centers (RC) of purple bacteria. Apart from opens in which electron can be transported in a natural way, also closed RCs, with electron transfer blocked at some point, were examined. Both wild type RCs and set of mutated RCs were investigated. The aim of selected mutations was to change free energy gap between P+BA- and P+HA-, where P is the primary electron donor and BA and HA are electron acceptors. The main goal of this paper was to construct recombination model consistent with the experimental data. The thesis is divided into two main parts: introduction, containing basic information about photosynthesis, photosynthetic pigments, purple bacteria, RCs, and materials and methods used in the experimental part. Results and outcomes are presented in the second part. The most important of them are direct observation of P+BA- state in wild type RC and multiexponential character of the decay of P+HA- state in closed RC, due to its relaxation. On the basis of the experimental results, free energy gap ΔG between P+BA- and P+HA states in charge separation time scale (few ps) as well as in charge recombination time scale (several hundred ps – several ns) were estimated and a self-consistent charge recombination model in closed RC was constructed. According to the proposed model, charge recombination occurs predominantly from the P+BA- state and the energy gap between P+HA- and P+BA- increases in time because of relaxation – passive response of protein to the appearance of electric charges. P+HA- decays in multiexponential manner because relaxation occurs on the same time scale as the charge recombination does