Search

Global analysis of a set of room temperature transient absorption spectra of Rhodobacter sphaeroidesreaction centers, recorded in wide temporal and spectral ranges and triggered by femtosecond excitation of accessory bacteriochlorophylls at 800 nm, is presented. The data give a comprehensive review of all spectral dynamics features in the visible and near UV, from 330 to 700 nm, related to the primary events in the Rb. sphaeroidesreaction center: excitation energy transfer from the accessory bacteriochlorophylls (B) to the primary donor (P), primary charge separation between the primary donor and primary acceptor (bacteriopheophytin, H), and electron transfer from the primary to the secondary electron acceptor (ubiquinone). In particular, engagement of the accessory bacteriochlorophyll in primary charge separation is shown as an intermediate electron acceptor, and the initial free energy gap of ∼40 meV, between the states P+BA−and P+HA−is estimated. The size of this gap is shown to be constant for the whole 230 ps lifetime of the P+HA−state. The ultrafast spectral dynamics features recorded in the visible range are presented against a background of results from similar studies performed for the last two decades. [ABSTRACT FROM AUTHOR]

We investigated the influence of a range of factors-temperature, redox midpoint potential of an electron carrier, and protein dynamics-on nanosecond electron transfer within a protein. The model reaction was back electron transfer from a bacteriopheophytin anion, H A - , to an oxidized primary electron donor, P + , in a wild type Rhodobacter sphaeroides reaction center (RC) with a permanently reduced secondary electron acceptor (quinone, Q A - ). Also used were two modified RCs with single amino acid mutations near the monomeric bacteriochlorophyll, B A , located between P and H A . Both mutant RCs showed significant slowing down of this back electron transfer reaction with decreasing temperature, similar to that observed with the wild type RC, but contrasting with a number of single point mutant RCs studied previously. The observed similarities and differences are explained in the framework of a (P + B A -  ↔ P + H A - ) equilibrium model with an important role played by protein relaxation. The major cause of the observed temperature dependence, both in the wild type RC and in the mutant proteins, is a limitation in access to the thermally activated pathway of charge recombination via the state P + B A - at low temperatures. The data indicate that in all RCs both charge recombination pathways, the thermally activated one and a direct one without involvement of the P + B A - state, are controlled by the protein dynamics. It is concluded that the modifications of the protein environment affect the overall back electron transfer kinetics primarily by changing the redox potential of B A and not by changing the protein relaxation dynamics., (© 2021. The Author(s).)

In 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.

The temperature dependence of charge recombination of the pair P(+)HA(-) in isolated reaction centers from the purple bacterium Rhodobacter sphaeroides with prereduced quinone QA was studied by sub-nanosecond to microsecond time-scale transient absorption. Overall, the kinetics slowed down substantially upon cooling from room temperature to ∼200 K, and then remained virtually unchanged down to 77 K, indicating the coexistence of two competitive pathways of charge recombination, a thermally-activated pathway appearing only above ~200 K and a temperature-independent pathway. In our modelling, the thermally activated pathway includes an uphill electron transfer from HA(-) to BA(-) leading to transient formation of the state P(+)BA(-), whereas the temperature-independent pathway is due to direct downhill electron transfer from HA(-) to P(+). At all temperatures studied, the kinetics could be approximated by a four-component decay. Detailed analysis of the lifetimes and amplitudes of particular phases over the range of temperatures suggests that the kinetically resolved phases reveal the consecutive appearance of three conformational states characterized by an increasing free energy gap between the states P(+)BA(-) and P(+)HA(-). The initial gap between these states was estimated to be only ~8 meV, the intermediate gap being ~92 meV, and the final gap ~135 meV, with no dependence on temperature. It was also calculated through a very straightforward approach that the relaxation process from the initial to the intermediate state occurs within 0.6 ± 0.1 ns, whereas the second step of relaxation from the intermediate to the final state takes 11 ± 2 ns. Both phases of the protein relaxation process are essentially temperature-independent. Possible alternative models to describe the experimental data that cannot be definitely excluded are also discussed.

Time-resolved spectroscopic studies of recombination of the P(+)HA(-) radical pair in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides give an opportunity to study protein dynamics triggered by light and occurring over the lifetime of P(+)HA(-). The state P(+)HA(-) is formed after the ultrafast light-induced electron transfer from the primary donor pair of bacteriochlorophylls (P) to the acceptor bacteriopheophytin (HA). In order to increase the lifetime of this state, and thus increase the temporal window for the examination of protein dynamics, it is possible to block forward electron transfer from HA(-) to the secondary electron acceptor QA. In this contribution, the dynamics of P(+)HA(-) recombination were compared at a range of temperatures from 77 K to room temperature, electron transfer from HA(-) to QA being blocked either by prereduction of QA or by genetic removal of QA. The observed P(+)HA(-) charge recombination was significantly slower in the QA-deficient RCs, and in both types of complexes, lowering the temperature from RT to 77 K led to a slowing of charge recombination. The effects are explained in the frame of a model in which charge recombination occurs via competing pathways, one of which is thermally activated and includes transient formation of a higher-energy state, P(+)BA(-). An internal electrostatic field supplied by the negative charge on QA increases the free energy levels of the state P(+)HA(-), thus decreasing its energetic distance to the state P(+)BA(-). In addition, the dielectric response of the protein environment to the appearance of the state P(+)HA(-) is accelerated from ∼50-100 ns in the QA-deficient mutant RCs to ∼1-16 ns in WT RCs with a negatively charged QA(-). In both cases, the temperature dependence of the protein dynamics is weak.

The kinetics of recombination of the P(+)H(A)(-) radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, ΔG, between the charge separated states P(+)B(A)(-) and P(+)H(A)(-) was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller ΔG than in the wild-type, and was slowed in a mutant having a larger ΔG. The free energy difference between the state P(+)H(A)(-) and the PH(A) ground state was unaffected by most of these mutations. These observations were consistent with a model in which the P(+)H(A)(-) → PH(A) charge recombination is thermally activated and occurs via the intermediate state P(+)B(A)(-), with a mean rate related to the size of the ΔG between the states P(+)B(A)(-) and P(+)H(A)(-) and not the ΔG between P(+)H(A)(-) and the ground state. A more detailed analysis of charge recombination in the mutants showed that the kinetics of the reaction were multiexponential, and characterized by ~0.5, ~1-3, and 7-17 ns lifetimes, similar to those measured for wild-type reaction centers. The exact lifetimes and relative amplitudes of the three components were strongly modulated by the mutations. Two models were considered in order to explain the observed multiexponentiality and modulation, involving heterogeneity or relaxation of P(+)H(A)(-) states, with the latter model giving a better fit to the experimental results.

Transient absorption spectroscopy with a time resolution of approximately 1 ns was applied to study the decay of the primary radical pair P+H- in Rhodobacter sphaeroides R-26 reaction centers with blocked electron transfer from H- to QA. The block in the electron transfer was realized in two ways: by either reducing or removing QA. We found very different kinetics of the P+H- decay in these two cases. Convolution of the multiexponential decay with the instrument response function allowed resolution of as many as three kinetic components of <1-, 3-4-, and 9-12-ns lifetimes in chromatophores with QA reduced and in isolated reaction centers both with QA either reduced or removed (with or without o-phenanthroline) but with variable relative amplitudes. Removing QA or adding o-phenanthroline to isolated reaction centers increased the amplitude of the slowest decay phase relative to that of the fastest phase. On the basis of these observations, we propose that reaction centers adopt three conformational states characterized by different decay kinetics of P+H-. These conformational states appear to be controlled by the charges in the vicinity of the QA site as revealed by the effects of QA reduction and o-phenanthroline-mediated protonation of the sites close to QA.