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Using molecular dynamics simulations and steady-state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far-UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature-dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.

We have measured the rate of the initial electron-transfer process as a function of temperature in reaction centers in a native strain of the photosynthetic bacterium Rhodobacter sphaeroides and two mutants generated by site-directed mutagenesis. In the mutants, a tyrosine residue in the vicinity of the primary electron donor and acceptor molecules was replaced by either phenylalanine or isoleucine. The electron-transfer reaction is slower in the mutants and has a qualitatively different dependence on temperature. In native reaction centers the rate increases as the temperature is reduced, in the phenylalanine mutant it is virtually independent of temperature, and in the isoleucine mutant it decreases with decreasing temperature. At 77 K, the electron-transfer reaction is approximately 30 times slower in the isoleucine mutant than in the native. These observations support the view that tyrosine-(M)210 plays an important role in the electron-transfer mechanism. In the isoleucine mutant at low temperatures, the stimulated emission from the excited reaction center undergoes a time-dependent shift to shorter wavelengths.

In bacterial photosynthesis it has been proposed that an electron is transferred from the excited singlet state (P∗) of theprimary electron donor first to a bacteriochlorophyll (BChl) molecule, generating a P + Chl charge-transfer state, and that BChl - subsequently (4–7 ps) transfers an electron to a bacteriopheophytin molecule. Here we review the evidence for and against the intermediacy of P + BChl - and present picosecond transient absorption and kinetic data that bear on this point. We conclude that there is no convincing evidence that P + BChl is a kinetically or spectrally resolved intermediary state in the charge separation process.

The reorganization energy (lambda) for electron transfer from the primary electron donor (P*) to the adjacent bacteriochlorophyll (B) in photosynthetic bacterial reaction centers is explored by molecular-dynamics simulations. Relatively long (40 ps) molecular-dynamics trajectories are used, rather than free energy perturbation techniques. When the surroundings of the reaction center are modeled as a membrane, lambda for P* B --> P+ B- is found to be approximately 1.6 kcal/mol. The results are not sensitive to the treatment of the protein's ionizable groups, but surrounding the reaction center with water gives higher values of lambda (approximately 6.5 kcal/mol). In light of the evidence that P+ B- lies slightly below P* in energy, the small lambda obtained with the membrane model is consistent with the speed and temperature independence of photochemical charge separation. The calculated reorganization energy is smaller than would be expected if the molecular dynamics trajectories had sampled the full conformational space of the system. Because the system does not relax completely on the time scale of electron transfer, the lambda obtained here probably is more pertinent than the larger value that would be obtained for a fully equilibrated system.

An assertion that the primary photochemistry of photosynthesis can violate the Second Law of thermodynamics in certain efficient systems has been put forward by Jennings et al., who maintain their position strongly despite an argument to the contrary by Lavergne. We identify a specific omission in the calculation of Jennings et al. and show that no violation of the Second Law occurs, regardless of the photosynthetic efficiency.

The rate of the electron-transfer reaction between bacteriopheophytin and the first quinone in isolated reaction centers of Rhodopseudomonas sphaeroides has an unusual temperature dependence. The rate increases about threefold with decreasing temperature between 300 and 25 K, and decreases abruptly at temperatures below 25 K. Partial deuteration of the reaction centers alters the temperature dependence of the rate constant. Qualitative features of the temperature dependence can be understood in the context of a theory of nonadiabatic electron transfer (Sarai, 1980. Biochim. Biophys. Acta 589:71–83). We conclude that very low-energy (10–50 cm-1) processes, perhaps skeletal vibrations of the protein, are important to electron transfer. Higher-energy vibrations, possibly involving the pyrrolic N--H bonds of bacteriopheophytin, also are important in this process.

Absorbance changes reflecting the formation of a transient radical-pair state, P F , were measured in reaction centers from Rhodopseudomonas sphaeroides under conditions that blocked electron transfer to a later carrier (a quinone, Q). The temperature dependence of the absorbance changes suggests that P F is an equilibrium mixture of two states, which appear to be mainly 1 [P [unk] B [unk] ] and 1 [P [unk] H [unk] ]. P is a bacteriochlorophyll dimer, B is a bacteriochlorophyll absorbing at 800 nm, and H is a bacteriopheophytin. In the presence of Q [unk] , the energy of 1 [P [unk] B [unk] ] is about 0.025 eV above that of 1 [P [unk] H [unk] ], 1 [P [unk] H [unk] ] can decay to a triplet state, P R , which also is an equilibrium mixture of two states, separated by about 0.03 eV. The lower of these appears to be mainly a locally excited triplet state of P, 3 P; the upper state contains a major contribution from a triplet charge-transfer state, 3 [P [unk] B [unk] ]. The temperature dependence of delayed fluorescence from P R indicates that 3 P lies 0.40 eV below the excited singlet state, P * , which is about 0.05 eV above 1 [P [unk] H [unk] ]. The 1,3 [P [unk] B [unk] ] charge-transfer states thus appear to interact with the locally excited states of P and B to give singlet and triplet states that are separated in energy by about 0.35 eV. This is 10 6 times larger than the splitting between 1 [P [unk] H [unk] ] and 3 [P [unk] H [unk] ] and implies strong orbital overlap between P [unk] and B [unk] . This is consistent with recent picosecond studies which suggest that electron transfer from P * to B occurs within 1 ps and is followed in 4 to 10 ps by electron transfer from B [unk] to H.

Preparations of photosynthetic reaction centers from Rhodopseudomonas sphaeroides were excited with flashes lasting approximately 8 psec. Immediately after the excitation, there appeared a transient state which was characterized by new absorption bands near 500 and 680 nm, by a bleaching of bands near 540, 600, 760, and 870 nm, and by a blue shift of a band near 800 nm. The transient state decayed with an exponential decay time,t, of 246 plus or minus 16 psec after the flash. As the transient state decayed, the radical cation of the reaction center bacteriochlorophyll complex appeared. This indicates that the transient state is an intermediate in the photooxidation of the bacteriochlorophyll. The absorpiton spectrum of the transient state shows the state to be identical with a state (P-F) which has been detected previously in reaction centers that are prevented from completing the photooxidation, because of chemical reduction of the electron acceptor. Analysis of the spectrum suggests that the formation of P-F involves electron transfer from one bacteriochlorophyll molecule to another within the reaction center, or possibly from bacteriochlorophyll to the bacteriopheophytin of the complex. The initial absorbance changes after flash excitation also include a bleaching of an absorption band at 800 nm. The bleaching decays with tau approximately equal to 30 pse. The bleaching appers not to be a secondary effect, but rather to revael another early step in the primary photochemical reaction.