Bacteriochlorophyll Excited-State Quenching Pathways in Bacterial Reaction Centers with the Primary Donor Oxidized

One striking feature of bacterial reaction centers is that while they show a high degree of structural symmetry, function is entirely asymmetric: excitation of the primary electron donor, P, a bacteriochlorophyll (BChl) dimer, results almost exclusively in electron transfer along one of the two symmetric electron transfer pathways. Here another functional asymmetry of the reaction center is explored; i.e., the two monomer BChl molecules (B_A and B_B) have distinct interactions with P in the oxidized state, P⁺. Previous work has suggested that the excited states of both B_A and B_B were quenched via energy transfer to P⁺ within a few hundred femtoseconds. Here, it is shown that various excitation wavelengths, corresponding to different initial B_A and B_B excited states, result in distinct reaction pathways, and which pathway dominates depends both on the initial excited state formed and on the electronic structure of P⁺. In particular, it is possible to specifically excite the Q_X transition of B_B by using excitation at 495 nm directly into the carotenoid S₂ state which then undergoes energy transfer to B_B. This results in the formation of a new state on the picosecond time scale that is both much longer lived and spectrally different than what one would expect for a simple excited state. Combining results from additional measurements using nonselective 600 or 800 nm excitation of both B_A and B_B to the Q_X or Q_Y states, respectively, it is found that B_B* and B_A* are quenched by P⁺ with different kinetics and mechanisms. B_A* formed using either Q_X or Q_Y excitation appears to decay rapidly (∼200 fs) without a detectable intermediate. In contrast, B_B* formed via Q_X excitation predominantly generates the long-lived state referred to above via an electron transfer reaction from the Q_X excited state of B_B to P⁺. This reaction is in competition with intramolecular relaxation of the Q_X state to the lowest singlet excited state. The Q_Y excited state of B_B appears to undergo the electron transfer reaction seen upon Q_X excitation only to a very limited extent and is largely quenched via energy transfer to P⁺. Finally, the ability of P⁺ to quench B_B* depends on the electronic structure of P⁺. The asymmetric charge distribution between the two halves of P in the native reaction center is effectively reversed in the mutant HF­(L168)/LH­(L131), and in this case, the rate of quenching decreases significantly

Utilizing the Dynamic Stark Shift as a Probe for Dielectric Relaxation in Photosynthetic Reaction Centers During Charge Separation

In photosynthetic reaction centers, the electric field generated by light-induced charge separation produces electrochromic shifts in the transitions of reaction center pigments. The extent of this Stark shift indirectly reflects the effective field strength at a particular cofactor in the complex. The dynamics of the effective field strength near the two monomeric bacteriochlorophylls (B_A and B_B) in purple photosynthetic bacterial reaction centers has been explored near physiological temperature by monitoring the time-dependent Stark shift during charge separation (dynamic Stark shift). This dynamic Stark shift was determined through analysis of femtosecond time-resolved absorbance change spectra recorded in wild type reaction centers and in four mutants at position M210. In both wild type and the mutants, the kinetics of the dynamic Stark shift differ from those of electron transfer, though not in the same way. In wild type, the initial electron transfer and the increase in the effective field strength near the active-side monomer bacteriochlorophyll (B_A) occur in synchrony, but the two signals diverge on the time scale of electron transfer to the quinone. In contrast, when tyrosine is replaced by aspartic acid at M210, the kinetics of the B_A Stark shift and the initial electron transfer differ, but transfer to the quinone coincides with the decay of the Stark shift. This is interpreted in terms of differences in the dynamics of the local dielectric environment between the mutants and the wild type. In wild type, comparison of the Stark shifts associated with B_A and B_B on the two quasi-symmetric halves of the reaction center structure confirm that the effective dielectric constants near these cofactors are quite different when the reaction center is in the state P⁺Q_A^–, as previously determined by Steffen et al. at 1.5 K (Steffen, M. A.; et al. Science 1994, 264, 810−816). However, it is not possible to determine from static, low-temperature measurments if the difference in the effective dielectric constant between the two sides of the reaction center is manifest on the time scale of initial electron transfer. By comparing directly the Stark shift dynamics of the ground-state spectra of the two monomer bacteriochlorophylls, it is evident that there is, in fact, a large dielectric difference between protein environments of the two quasi-symmetric electron-transfer branches on the time scale of initial electron transfer and that the effective dielectric constant in the region continues to evolve on a time scale of hundreds of picoseconds

Interenzyme Substrate Diffusion for an Enzyme Cascade Organized on Spatially Addressable DNA Nanostructures

Spatially addressable DNA nanostructures facilitate the self-assembly of heterogeneous elements with precisely controlled patterns. Here we organized discrete glucose oxidase (GOx)/horseradish peroxidase (HRP) enzyme pairs on specific DNA origami tiles with controlled interenzyme spacing and position. The distance between enzymes was systematically varied from 10 to 65 nm, and the corresponding activities were evaluated. The study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Strongly enhanced activity was observed for those assemblies in which the enzymes were closely spaced, while the activity dropped dramatically for enzymes as little as 20 nm apart. Increasing the spacing further resulted in a much weaker distance dependence. Combined with diffusion modeling, the results suggest that Brownian diffusion of intermediates in solution governed the variations in activity for more distant enzyme pairs, while dimensionally limited diffusion of intermediates across connected protein surfaces contributed to the enhancement in activity for closely spaced GOx/HRP assemblies. To further test the role of limited dimensional diffusion along protein surfaces, a noncatalytic protein bridge was inserted between GOx and HRP to connect their hydration shells. This resulted in substantially enhanced activity of the enzyme pair

Exploring Peptide Space for Enzyme Modulators

A method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A polyvinyl alcohol solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide spot. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase, alkaline phosphatase, and β-galactosidase and substantially altered enzyme activity by comparing the binding level of peptide to enzyme and bound enzyme activity. This basic technique may be generally applicable to find peptides or other small molecules that modify enzyme activity

Feature-Level MALDI-MS Characterization of in Situ-Synthesized Peptide Microarrays

Characterizing the chemical composition of microarray features is a difficult yet important task in the production of in situ-synthesized microarrays. Here, we describe a method to determine the chemical composition of microarray features, directly on the feature. This method utilizes nondiffusional chemical cleavage from the surface along with techniques from MALDI-MS tissue imaging, thereby making the chemical characterization of high-density microarray features simple, accurate, and amenable to high-throughput

Electron Transfer in Bacterial Reaction Centers with the Photoactive Bacteriopheophytin Replaced by a Bacteriochlorophyll through Coordinating Ligand Substitution

The influence of amino acid substitutions at position M214 (M-subunit, residue 214) on the rate and pathway of electron transfer involving the bacteriopheophytin cofactor, HA, in a bacterial photosynthetic reaction center has been explored in a series of Rhodobacter sphaeroides mutants. The M214 leucine (L) residue of the wild type was replaced with histidine (H), glutamine (Q), and asparagine (N), creating the mutants M214LH, M214LQ, and M214LN, respectively. As has been reported previously for M214LH, each of these mutations resulted in a bacteriochlorophyll molecule in place of a bacteriopheophytin in the HA pocket, forming so-called β-type mutants (in which the HA cofactor is called βA). In addition, these mutations changed the properties of the surrounding protein environment in terms of charge distribution and the amino acid side chain volume. Electron transfer reactions from the excited primary donor P to the acceptor QA were characterized using ultrafast transient absorption spectroscopic techniques. Similar to that of the previously characterized M214LH (β mutant), the strong energetic mixing of the P+BA– and P+βA– states (the mixed anion is denoted I–) increased the rate of charge recombination between P+ and I– in competition with the I– → QA forward reaction. This reduced the overall yield of charge separation forming the P+QA– state. While the kinetics of the primary electron transfer forming P+I– were essentially identical in all three β mutants, the rates of the βA– (I–) → QA electron transfer in M214LQ and M214LH were very similar but quite different from that of the M214LN mutant. The observed yield changes and the differences in kinetics are correlated more closely with the volume of the mutated amino acid than with their charge characteristics. These results are consistent with those of previous studies of a series of M214 mutants with different sizes of amino acid side chains that did not alter the HA cofactor composition [Pan, J., et al. (2013) J. Phys. Chem. B 117, 7179–7189]. Both studies indicate that protein relaxation in this region of the reaction center plays a key role in stabilizing charge-separated states involving the HA or βA cofactor. The effect is particularly pronounced for reactions occurring on time scales of tens and hundreds of picoseconds (forward transfer to the QA and charge recombination)

Increasing the Rate of Energy Transfer between the LHI Antenna and the Reaction Center in the Photosynthetic Bacterium <i>Rhodobacter </i><i>s</i><i>phaeroides</i>

Energy transfer from Rhodobacter sphaeroides light-harvesting complex I (LHI) to the reaction center (RC) was investigated with steady-state and time-resolved fluorescence spectroscopy. Chromatophores isolated from a strain containing LHI with the mutation αTrp43 to Phe (LHI mutant) and strains containing either the LHI mutant with wild-type RCs (LHI mutant + WT RC) or the LHI mutant with the RC mutations LH(L131)+LH(M160)+FH(M197) (LHI mutant + T1 RC) were investigated at 294 and 77 K. In the LHI mutant, absorption and fluorescence spectra were blue-shifted by 21 nm compared to wild-type LHI. The energy transfer from mutated LHI to the RC occurs about two times faster than energy transfer from wild-type LHI to the RC. The acceleration of energy transfer is consistent with the increase in the energy transfer rate estimated from the spectral overlap between the RC absorbance and the LHI fluorescence according to Förster energy transfer theory

Comparing the Temperature Dependence of Photosynthetic Electron Transfer in Chloroflexus aurantiacus and Rhodobactor sphaeroides Reaction Centers

The process of electron transfer from the special pair, P, to the primary electron donor, HA, in quinone-depleted reaction centers (RCs) of Chloroflexus (Cf.) aurantiacus has been investigated over the temperature range from 10 to 295 K using time-resolved pump–probe spectroscopic techniques. The kinetics of the electron transfer reaction, P* → P+HA –, was found to be nonexponential, and the degree of nonexponentiality increased strongly as temperature decreased. The temperature-dependent behavior of electron transfer in Cf. aurantiacus RCs was compared with that of the purple bacterium Rhodobacter (Rb.) sphaeroides. Distinct transitions were found in the temperature-dependent kinetics of both Cf. aurantiacus and Rb. sphaeroides RCs, at around 220 and 160 K, respectively. Structural differences between these two RCs, which may be associated with those differences, are discussed. It is suggested that weaker protein–cofactor hydrogen bonding, stronger electrostatic interactions at the protein surface, and larger solvent interactions likely contribute to the higher transition temperature in Cf. aurantiacus RCs temperature-dependent kinetics compared with that of Rb. sphaeroides RCs. The reaction-diffusion model provides an accurate description for the room-temperature electron transfer kinetics in Cf. aurantiacus RCs with no free parameters, using coupling and reorganization energy values previously determined for Rb. sphaeroides, along with an experimental measure of protein conformational diffusion dynamics and an experimental literature value of the free energy gap between P* and P+HA –

Orange Carotenoid Protein as a Control Element in an Antenna System Based on a DNA Nanostructure

Taking inspiration from photosynthetic mechanisms in natural systems, we introduced a light-sensitive photo protective quenching element to an artificial light-harvesting antenna model to control the flow of energy as a function of light intensity excitation. The orange carotenoid protein (OCP) is a nonphotochemical quencher in cyanobacteria: under high-light conditions, the protein undergoes a spectral shift, and by binding to the phycobilisome, it absorbs excess light and dissipates it as heat. By the use of DNA as a scaffold, an antenna system made of organic dyes (Cy3 and Cy5) was constructed, and OCP was assembled on it as a modulated quenching element. By controlling the illumination intensity, it is possible to switch the direction of excitation energy transfer from the donor Cy3 to either of two acceptors. Under low-light conditions, energy is transferred from Cy3 to Cy5, and under intense illumination, energy is partially transferred to OCP as well. These results demonstrate the feasibility of controlling the pathway of energy transfer using light intensity in an engineered light-harvesting system

Ultrafast Electron Transfer Kinetics in the LM Dimer of Bacterial Photosynthetic Reaction Center from <i>Rhodobacter sphaeroides</i>

It has become increasingly clear that dynamics plays a major role in the function of many protein systems. One system that has proven particularly facile for studying the effects of dynamics on protein-mediated chemistry is the bacterial photosynthetic reaction center from <i>Rhodobacter sphaeroides</i>. Previous experimental and computational analysis have suggested that the dynamics of the protein matrix surrounding the primary quinone acceptor, Q_A, may be particularly important in electron transfer involving this cofactor. One can substantially increase the flexibility of this region by removing one of the reaction center subunits, the H-subunit. Even with this large change in structure, photoinduced electron transfer to the quinone still takes place. To evaluate the effect of H-subunit removal on electron transfer to Q_A, we have compared the kinetics of electron transfer and associated spectral evolution for the LM dimer with that of the intact reaction center complex on picosecond to millisecond time scales. The transient absorption spectra associated with all measured electron transfer reactions are similar, with the exception of a broadening in the Q_X transition and a blue-shift in the Q_Y transition bands of the special pair of bacteriochlorophylls (P) in the LM dimer. The kinetics of the electron transfer reactions not involving quinones are unaffected. There is, however, a 4-fold decrease in the electron transfer rate from the reduced bacterio­pheophytin to Q_A in the LM dimer compared to the intact reaction center and a similar decrease in the recombination rate of the resulting charge-separated state (P⁺Q_A^–). These results are consistent with the concept that the removal of the H-subunit results in increased flexibility in the region around the quinone and an associated shift in the reorganization energy associated with charge separation and recombination