Bound manganese oxides capable of reducing the bacteriochlorophyll dimer of modified reaction centers from Rhodobacter sphaeroides.

A biohybrid model system is described that interfaces synthetic Mn-oxides with bacterial reaction centers to gain knowledge concerning redox reactions by metal clusters in proteins, in particular the Mn4CaO5 cluster of photosystem II. The ability of Mn-oxides to bind to modified bacterial reaction centers and transfer an electron to the light-induced oxidized bacteriochlorophyll dimer, P+, was characterized using optical spectroscopy. The environment of P was altered to obtain a high P/P+ midpoint potential. In addition, different metal-binding sites were introduced by substitution of amino acid residues as well as extension of the C-terminus of the M subunit with the C-terminal region of the D1 subunit of photosystem II. The Mn-compounds MnO2, αMn2O3, Mn3O4, CaMn2O4, and Mn3(PO4)2 were tested and compared to MnCl2. In general, addition of the Mn-compounds resulted in a decrease in the amount of P+ while the reduced quinone was still present, demonstrating that the Mn-compounds can serve as secondary electron donors. The extent of P+ reduction for the Mn-oxides was largest for αMn2O3 and CaMn2O4 and smallest for Mn3O4 and MnO2. The addition of Mn3(PO4)2 resulted in nearly complete P+ reduction, similar to MnCl2. Overall, the activity was correlated with the initial oxidation state of the Mn-compound. Transient optical measurements showed a fast kinetic component, assigned to reduction of P+ by the Mn-oxide, in addition to a slow component due to charge recombination. The results support the conjecture that the incorporation of Mn-oxides by ancient anoxygenic phototrophs was a step in the evolution of oxygenic photosynthesis.

Influence of Hydrogen Bonds on the Electron-Phonon Coupling Strength/Marker Mode Structure and Charge Separation Rates in Reaction Centers from Rhodobacter sphaeroides.

Low-temperature persistent and transient hole-burning (HB) spectra are presented for the triple hydrogen-bonded L131LH + M160LH + M197FH mutant of Rhodobacter sphaeroides. These spectra expose the heterogeneous nature of the P-, B-, and H-bands, consistent with a distribution of electron transfer (ET) times and excitation energy transfer (EET) rates. Transient P+QA- holes are observed for fast (tens of picoseconds or faster) ET times and reveal strong coupling to phonons and marker mode(s), while the persistent holes are bleached in a fraction of reaction centers with long-lived excited states characterized by much weaker electron-phonon coupling. Exposed differences in electron-phonon coupling strength, as well as a different coupling to the marker mode(s), appear to affect the ET times. Both resonantly and nonresonantly burned persistent HB spectra show weak blue- (∼150 cm-1) and large, red-shifted (∼300 cm-1) antiholes of the P band. Slower EET times from the H- and B-bands to the special pair dimer provide new insight on the influence of hydrogen bonds on mutation-induced heterogeneity.

Influence of the Electrochemical Properties of the Bacteriochlorophyll Dimer on Triplet Energy-Transfer Dynamics in Bacterial Reaction Centers.

Energetics, protein dynamics, and electronic coupling are the key factors in controlling both electron and energy transfer in photosynthetic bacterial reaction centers (RCs). Here, we examine the rates and mechanistic pathways of the P+HA- radical-pair charge recombination, triplet state formation, and subsequent triplet energy transfer from the triplet state of the bacteriochlorophyll dimer (P) to the carotenoid in a series of mutant RCs (L131LH + M160LH (D1), L131LH + M197FH (D2), and L131LH + M160LH + M197FH (T1)) of Rhodobacter sphaeroides. In these mutants, the electronic structure of P is perturbed and the P/P+ midpoint potential is systematically increased due to addition of hydrogen bonds between P and the introduced residues. High-resolution, broad-band, transient absorption spectroscopy on the femtosecond to microsecond timescale shows that the charge recombination rate increases and the triplet energy transfer rate decreases in these mutants relative to the wild type (WT). The increase of the charge recombination rate is correlated to the increase in the energy level of P+HA- and the increase in the P/P+ midpoint potential. On the other hand, the decrease in rate of triplet energy transfer in the mutants can be explained in terms of a lower energy of 3P and a shift in the electron spin density distribution in the bacteriochlorophylls of P. The triplet energy-transfer rate follows the order of WT > L131LH + M197FH > L131LH + M160LH > L131LH + M160LH + M197FH, both at room temperature and at 77 K. A pronounced temperature dependence of the rate is observed for all of the RC samples. The activation energy associated to this process is increased in the mutants relative to WT, consistent with a lower 3P energy due to the addition of hydrogen bonds between P and the introduced residues.

Binding and Energetics of Electron Transfer between an Artificial Four-Helix Mn-Protein and Reaction Centers from Rhodobacter sphaeroides.

The ability of an artificial four-helix bundle Mn-protein, P1, to bind and transfer an electron to photosynthetic reaction centers from the purple bacterium Rhodobacter sphaeroides was characterized using optical spectroscopy. Upon illumination of reaction centers, an electron is transferred from P, the bacteriochlorophyll dimer, to QA, the primary electron acceptor. The P1 Mn-protein can bind to the reaction center and reduce the oxidized bacteriochlorophyll dimer, P+, with a dissociation constant of 1.2 µM at pH 9.4, comparable to the binding constant of c-type cytochromes. Amino acid substitutions of surface residues on the Mn-protein resulted in increases in the dissociation constant to 8.3 µM. The extent of reduction of P+ by the P1 Mn-protein was dependent on the P/P+ midpoint potential and the pH. Analysis of the free energy difference yielded a midpoint potential of approximately 635 mV at pH 9.4 for the Mn cofactor of the P1 Mn-protein, a value similar to those found for other Mn cofactors in proteins. The linear dependence of -56 mV/pH is consistent with one proton being released upon Mn oxidation, allowing the complex to maintain overall charge neutrality. These outcomes demonstrate the feasibility of designing four-helix bundles and other artificial metalloproteins to bind and transfer electrons to bacterial reaction centers and establish the usefulness of this system as a platform for designing sites to bind novel metal cofactors capable of performing complex oxidation-reduction reactions.

Biochemical and spectroscopic characterization of dinuclear Mn-sites in artificial four-helix bundle proteins.

To better understand metalloproteins with Mn-clusters, we have designed artificial four-helix bundles to have one, two, or three dinuclear metal centers able to bind Mn(II). Circular dichroism measurements showed that the Mn-proteins have substantial α-helix content, and analysis of electron paramagnetic resonance spectra is consistent with the designed number of bound Mn-clusters. The Mn-proteins were shown to catalyze the conversion of hydrogen peroxide into molecular oxygen. The loss of hydrogen peroxide was dependent upon the concentration of protein with bound Mn, with the proteins containing multiple Mn-clusters showing greater activity. Using an oxygen sensor, the oxygen concentration was found to increase with a rate up to 0.4µM/min, which was dependent upon the concentrations of hydrogen peroxide and the Mn-protein. In addition, the Mn-proteins were shown to serve as electron donors to bacterial reaction centers using optical spectroscopy. Similar binding of the Mn-proteins to reaction centers was observed with an average dissociation constant of 2.3µM. The Mn-proteins with three metal centers were more effective at this electron transfer reaction than the Mn-proteins with one or two metal centers. Thus, multiple Mn-clusters can be incorporated into four-helix bundles with the capability of performing catalysis and electron transfer to a natural protein.

Mechanism of Triplet Energy Transfer in Photosynthetic Bacterial Reaction Centers.

In purple bacterial reaction centers, triplet excitation energy transfer occurs from the primary donor P, a bacteriochlorophyll dimer, to a neighboring carotenoid to prevent photodamage from the generation of reactive oxygen species. The BB bacteriochlorophyll molecule that lies between P and the carotenoid on the inactive electron transfer branch is involved in triplet energy transfer between P and the carotenoid. To expand the high-resolution spectral and kinetic information available for describing the mechanism, we investigated the triplet excited state formation and energy transfer pathways in the reaction center of Rhodobacter sphaeroides using pump-probe transient absorption spectroscopy over a broad spectral region on the nanosecond to microsecond time scale at both room temperature and at 77 K. Wild-type reaction centers were compared with a reaction center mutant (M182HL) in which BB is replaced by a bacteriopheophytin (Φ), as well as to reaction centers that lack the carotenoid. In wild-type reaction centers, the triplet energy transfer efficiency from P to the carotenoid was essentially unity at room temperature and at 77 K. However, in the M182HL mutant reaction centers, both the rate and efficiency of triplet energy transfer were decreased at room temperature, and at 77 K, no triplet energy transfer was observed, attributable to a higher triplet state energy of the bacteriopheophytin that replaces bacteriochlorophyll in this mutant. Finally, detailed time-resolved spectral analysis of P, carotenoid, and BB (Φ in the M182HL mutant) reveals that the triplet state of the carotenoid is coupled fairly strongly to the bridging intermediate BB in wild-type and Φ in the M182HL mutant, a fact that is probably responsible for the lack of any obvious intermediate 3BB/3Φ transient formation during triplet energy transfer.

Design of dinuclear manganese cofactors for bacterial reaction centers.

A compelling target for the design of electron transfer proteins with novel cofactors is to create a model for the oxygen-evolving complex, a Mn4Ca cluster, of photosystem II. A mononuclear Mn cofactor can be added to the bacterial reaction center, but the addition of multiple metal centers is constrained by the native protein architecture. Alternatively, metal centers can be incorporated into artificial proteins. Designs for the addition of dinuclear metal centers to four-helix bundles resulted in three artificial proteins with ligands for one, two, or three dinuclear metal centers able to bind Mn. The three-dimensional structure determined by X-ray crystallography of one of the Mn-proteins confirmed the design features and revealed details concerning coordination of the Mn center. Electron transfer between these artificial Mn-proteins and bacterial reaction centers was investigated using optical spectroscopy. After formation of a light-induced, charge-separated state, the experiments showed that the Mn-proteins can donate an electron to the oxidized bacteriochlorophyll dimer of modified reaction centers, with the Mn-proteins having additional metal centers being more effective at this electron transfer reaction. Modeling of the structure of the Mn-protein docked to the reaction center showed that the artificial protein likely binds on the periplasmic surface similarly to cytochrome c2, the natural secondary donor. Combining reaction centers with exogenous artificial proteins provides the opportunity to create ligands and investigate the influence of inhomogeneous protein environments on multinuclear redox-active metal centers. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Copper Environment in Artificial Metalloproteins Probed by Electron Paramagnetic Resonance Spectroscopy.

The design of binding sites for divalent metals in artificial proteins is a productive platform for examining the characteristics of metal-ligand interactions. In this report, we investigate the spectroscopic properties of small peptides and four-helix bundles that bind Cu(II). Three small peptides, consisting of 15 amino acid residues, were designed to have two arms, each containing a metal-binding site comprised of different combinations of imidazole and carboxylate side chains. Two four-helix bundles each had a binding site for a central dinuclear metal cofactor, with one design incorporating additional potential metal ligands at two identical sites. The small peptides displayed pH-dependent, metal-induced changes in the circular dichroism spectra, consistent with large changes in the secondary structure upon metal binding, while the spectra of the four-helix bundles showed a predominant α-helix content but only small structural changes upon metal binding. Electron paramagnetic resonance spectra were measured at X-band revealing classic Cu(II) axial patterns with hyperfine coupling peaks for the small peptides and four-helix bundles exhibiting a range of values that were related to the specific chemical natures of the ligands. The variety of electronic structures allow us to define the distinctive environment of each metal-binding site in these artificial systems, including the designed additional binding sites in one of the four-helix bundles.

Interface for Light-Driven Electron Transfer by Photosynthetic Complexes Across Block Copolymer Membranes.

Incorporation of membrane proteins into nanodevices to mediate recognition and transport in a collective and scalable fashion remains a challenging problem. We demonstrate how nanoscale photovoltaics could be designed using robust synthetic nanomembranes with incorporated photosynthetic reaction centers (RCs). Specifically, RCs from Rhodobacter sphaeroides are reconstituted spontaneously into rationally designed polybutadiene membranes to form hierarchically organized proteopolymer membrane arrays via a charge-interaction-directed reconstitution mechanism. Once incorporated, the RCs are fully active for prolonged periods based upon a variety of spectroscopic measurements, underscoring preservation of their 3D pigment configuration critical for light-driven charge transfer. This result provides a strategy to construct solar conversion devices using structurally versatile proteopolymer membranes with integrated RC functions to harvest broad regions of the solar spectrum.

Light-driven oxygen production from superoxide by Mn-binding bacterial reaction centers.

One of the outstanding questions concerning the early Earth is how ancient phototrophs made the evolutionary transition from anoxygenic to oxygenic photosynthesis, which resulted in a substantial increase in the amount of oxygen in the atmosphere. We have previously demonstrated that reaction centers from anoxygenic photosynthetic bacteria can be modified to bind a redox-active Mn cofactor, thus gaining a key functional feature of photosystem II, which contains the site for water oxidation in cyanobacteria, algae, and plants [Thielges M, et al. (2005) Biochemistry 44:7389-7394]. In this paper, the Mn-binding reaction centers are shown to have a light-driven enzymatic function; namely, the ability to convert superoxide into molecular oxygen. This activity has a relatively high efficiency with a k(cat) of approximately 1 s(-1) that is significantly larger than typically observed for designed enzymes, and a K(m) of 35-40 µM that is comparable to the value of 50 µM for Mn-superoxide dismutase, which catalyzes a similar reaction. Unlike wild-type reaction centers, the highly oxidizing reaction centers are not stable in the light unless they have a bound Mn. The stability and enzymatic ability of this type of Mn-binding reaction centers would have provided primitive phototrophs with an environmental advantage before the evolution of organisms with a more complex Mn(4)Ca cluster needed to perform the multielectron reactions required to oxidize water.

Carotenoid excited-state properties in photosynthetic purple bacterial reaction centers: effects of the protein environment.

Carotenoid excited-state properties are characterized and compared in reaction centers (RCs) of wild-type (WT) Rhodobacter (Rb.) sphaeroides , and a mutant VR(L157), in which the near-infrared absorbance band associated with the primary electron donor, P, is missing. Energy transfer from the carotenoid (spheroidenone) S(2) and relaxed S(1) excited states to an adjacent monomeric-bacteriochlorophyll is unchanged between WT and the mutant RC samples. However, two other excited states, including a vibrationally hot S(1) state and a state referred to as S*, have distinct properties in the two RCs. The lifetime of the hot S(1) state is significantly shortened in the P-less mutant compared to WT RCs (450 fs vs 800 fs, respectively), and there is a nearly 2-fold decrease in the efficiency of energy transfer from the carotenoid to bacteriochlorophyll in the P-less mutant relative to WT RCs. The fact that both the observed hot S(1) excited state lifetime and the energy transfer efficiency decrease in the mutant implies that the intrinsic lifetime of the hot S(1) state in the P-less mutant has decreased. Interestingly, the S* state is observed only in the P-less mutant, and it is not present in the WT. The change in the hot S(1) lifetime between WT and mutant RCs, and the formation of the S* state only in the mutant, suggests that the carotenoid binding pocket in the P-less mutant is substantially altered. The excited-state behavior of spheroidene in WT RCs isolated from anaerobically grown cells was also characterized and compared with previous studies of spheroidene in the light-harvesting complex II (LH2) from Rb. sphaeroides . Differences in the photophysical properties of spheroidene between WT RCs and LH2 parallel those observed for spheroidenone between WT and VR(L157) mutant RCs. On the basis of the structural information available for both RCs and LH2, it appears that the hot S(1) state and the S* state are sensitive to the structural constraints imposed by protein-carotenoid interactions. Finally, in the VR(L157) mutant, it is possible to directly observe the carotenoid triplet state, likely formed via quenching of the bacteriochlorophyll triplet state. This provides direct experimental evidence for triplet energy transfer to the carotenoid, a process that is integral to the photoprotective role of carotenoids in bacterial RCs.

Light-induced conformational changes in photosynthetic reaction centers: redox-regulated proton pathway near the dimer.

The influence of the hydrogen bonds on the light-induced structural changes were studied in the wild type and 11 mutants with different hydrogen bonding patterns of the primary electron donor of reaction centers from Rhodobacter sphaeroides. Previously, using the same set of mutants at pH 8, a marked light-induced change of the local dielectric constant in the vicinity of the dimer was reported in wild type and in mutants retaining Leu L131 that correlated with the recovery kinetics of the charge-separated state [ Deshmukh et al. (2011) Biochemistry, 50, 340-348]. In this work after prolonged illumination the recovery of the oxidized dimer was found to be multiphasic in all mutants. The fraction of the slowest phase, assigned to a recovery from a conformationally altered state, was strongly pH dependent and found to be extremely long at room temperature, at pH 6, with rate constants of ∼10(-3) s(-1). In wild type and in mutants with Leu at L131 the very long recovery kinetics was coupled to a large proton release at pH 6 and a decrease of up to 79 mV of the oxidation potential of the dimer. In contrast, in the mutants carrying the Leu to His mutation at the L131 position, only a negligible fraction of the dimer exhibited lowered potential, the large proton release was not observed, the oxidized dimer recovered 1 or 2 orders of magnitude faster depending on the pH, and the very long-lived state was not or barely detectable. These results are modeled as arising from the loss of a proton pathway from the bacteriochlorophyll dimer to the solvent when His is present at the L131 position.

Light-induced conformational changes in photosynthetic reaction centers: dielectric relaxation in the vicinity of the dimer.

Conformational changes near the bacteriochlorophyll dimer induced by continuous illumination were identified in the wild type and 11 different mutants of reaction centers from Rhodobacter sphaeroides. The properties of the bacteriochlorophyll dimer, which has a different hydrogen bonding pattern with the surrounding protein in each mutant, were characterized by steady-state and transient optical spectroscopy. After illumination for 1 min, in the absence of the secondary quinone, the recovery of the charge-separated states was nearly 1 order of magnitude slower in one group of mutants including the wild type than in the mutants carrying the Leu to His mutation at the L131 position. The slower recovery was accompanied by a substantial decrease in the electrochromic absorption changes associated with the Q(y) bands of the nearby monomers during the illumination. The other set of mutants containing the Leu L131 to His substitution exhibited slightly altered electrochromic changes that decreased only half as much during the illumination as in the other family of mutants. The correlation between the recovery of the charge-separated states in the light-induced conformation and the electrochromic absorption changes suggests a dielectric relaxation of the protein that stabilizes the charge on the dimer.

Effect of anions on the binding and oxidation of divalent manganese and iron in modified bacterial reaction centers.

The influence of different anions on the binding and oxidation of manganous and ferrous cations was studied in four mutants of bacterial reaction centers that can bind and oxidize these metal ions. Light-minus-dark difference optical and electron paramagnetic resonance spectroscopies were applied to monitor electron transfer from bound divalent metal ions to the photo-oxidized bacteriochlorophyll dimer in the presence of five different anions. At pH 7, bicarbonate was found to be the most effective for both manganese and iron binding, with dissociation constants around 1 muM in three of the mutants. The pH dependence of the dissociation constants for manganese revealed that only bicarbonate and acetate were able to facilitate the binding and oxidation of the metal ion between pH 6 and 8 where the tight binding in their absence could not otherwise be established. The data are consistent with two molecules of bicarbonate or one molecule of acetate binding to the metal binding site. For ferrous ion, the binding and oxidation was facilitated not only by bicarbonate and acetate, but also by citrate. Electron paramagnetic resonance spectra suggest differences in the arrangement of the iron ligands in the presence of the various anions.

Unusual temperature dependence of photosynthetic electron transfer due to protein dynamics.

The initial electron transfer rate and protein dynamics in wild type and five mutant reaction centers from Rhodobacter sphaeroides have been studied as a function of temperature (10-295 K). Detailed kinetic measurements of initial electron transfer in Rhodobacter sphaeroides reaction centers can be quantitatively described by a reaction diffusion formalism at all temperatures from 10 to 295 K. In this model, the time course of electron transfer is determined by the ability of the protein to interconvert between conformations until one is found where the activation energy for electron transfer is near zero. In reaction centers with a free energy for electron transfer similar to wild type, the reaction proceeds at least as fast at cryogenic temperatures as at room temperature. This may be because interconversion between conformations at low temperature is restricted to conformations with near zero activation energy (it is not possible to diffuse away from this region of conformational space). In contrast, mutants with a decreased free energy initially find themselves in conformations unfavorable for electron transfer and require more extensive conformational diffusion to achieve a low activation energy conformation. They therefore undergo electron transfer more slowly at 10 K vs 295 K.

Comparison of bacterial reaction centers and photosystem II.

In photosynthetic organisms, the utilization of solar energy to drive electron and proton transfer reactions across membranes is performed by pigment-protein complexes including bacterial reaction centers (BRCs) and photosystem II. The well-characterized BRC has served as a structural and functional model for the evolutionarily-related photosystem II for many years. Even though these complexes transfer electrons and protons across cell membranes in analogous manners, they utilize different secondary electron donors. Photosystem II has the unique ability to abstract electrons from water, while BRCs use molecules with much lower potentials as electron donors. This article compares the two complexes and reviews the factors that give rise to the functional differences. Also discussed are the modifications that have been performed on BRCs so that they perform reactions, such as amino acid and metal oxidation, which occur in photosystem II.

Protein dynamics control the kinetics of initial electron transfer in photosynthesis.

The initial electron transfer dynamics during photosynthesis have been studied in Rhodobacter sphaeroides reaction centers from wild type and 14 mutants in which the driving force and the kinetics of charge separation vary over a broad range. Surprisingly, the protein relaxation kinetics, as measured by tryptophan absorbance changes, are invariant in these mutants. By applying a reaction-diffusion model, we can fit the complex electron transfer kinetics of each mutant quantitatively, varying only the driving force. These results indicate that initial photosynthetic charge separation is limited by protein dynamics rather than by a static electron transfer barrier.

Environmental control of primary photochemistry in a mutant bacterial reaction center.

The core structure of the photosynthetic reaction center is quasisymmetric with two potential pathways (called A and B) for transmembrane electron transfer. Both the pathway and products of light-induced charge separation depend on local electrostatic interactions and the nature of the excited states generated at early times in reaction centers isolated from Rhodobacter sphaeroides. Here transient absorbance measurements were recorded following specific excitation of the Q(y)() transitions of P (the special pair of bacteriochlorophylls), the monomer bacteriochlorophylls (B(A) and B(B)), or the bacteriopheophytins (H(A) and H(B)) as a function of both buffer pH and detergent in a reaction center mutant with the mutations L168 His to Glu and L170 Asn to Asp in the vicinity of P and B(B). At a low pH in any detergent, or at any pH in a nonionic detergent (Triton X-100), the photochemistry of this mutant is faster than, but similar to, wild type (i.e. electron transfer occurs along the A-side, 390 nm excitation is capable of producing short-lived B-side charge separation (B(B)(+)H(B)(-)) but no long-lived B(B)(+)H(B)(-) is observed). Certain buffering conditions result in the stabilization of the B-side charge separated state B(B)(+)H(B)(-), including high pH in the zwitterionic detergent LDAO, even following excitation with low energy photons (800 or 740 nm). The most striking result is that conditions giving rise to stable B-side charge separation result in a lack of A-side charge separation, even when P is directly excited. The mechanism that links B(B)(+)H(B)(-) stabilization to this change in the photochemistry of P in the mutant is not understood, but clearly these two processes are linked and highly sensitive to the local electrostatic environment produced by buffering conditions (pH and detergent).