Effects of mutations near the bacteriochlorophylls in reaction centers from Rhodobacter sphaeroides.

Mutations were made in four residues near the bacteriochlorophyll cofactors of the photosynthetic reaction center from Rhodobacter sphaeroides. These mutations, L131 Leu to His and M160 Leu to His, near the dimer bacteriochlorophylls, and M203 Gly to Asp and L177 Ile to Asp, near the monomer bacteriochlorophylls, were designed to result in the placement of a hydrogen bond donor group near the ring V keto carbonyl of each bacteriochlorophyll. Perturbations of the electronic structures of the bacteriochlorophylls in the mutants are indicated by additional resolved transitions in the bacteriochlorophyll absorption bands in steady-state low-temperature and time-resolved room temperature spectra in three of the resulting mutant reaction centers. The major effect of the two mutations near the dimer was an increase up to 80 mV in the donor oxidation-reduction midpoint potential. Correspondingly, the calculated free energy difference between the excited state of the primary donor and the initial charge separated state decreased by up to 55 mV, the initial forward electron-transfer rate was up to 4 times slower, and the rate of charge recombination between the primary quinone and the donor was approximately 30% faster in these two mutants compared to the wild type. The two mutations near the monomer bacteriochlorophylls had minor changes of 25 mV or less in the donor oxidation-reduction potential, but the mutation close to the monomer bacteriochlorophyll on the active branch resulted in a roughly 3-fold decrease in the rate of the initial electron transfer.     

The structure and function of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>2</Subscript>: reaction center electron transfer complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c₂ (cyt c₂) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c₂ (cyt c₂) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c₂ is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers. Response under conditions of single electron turnover.

Light-induced electric current and potential responses have been measured across planar phospholipid membranes containing reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides. Under conditions in which the reaction centers are restricted to a single electron turnover, the responses can be correlated with the light-induced electron transfer reactions associated with the reaction center. The results indicate that electron transfer from the bacteriochlorophyll dimer to the primary ubiquinone molecule, and from ferrocytochrome c to the oxidized dimer occur in series across the planar membrane. Electron transfer from the primary to secondary ubiquinone molecule is not electrogenic.     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

Effect of deuteration and cryosolvents on the energy transduction in primary processes of photosynthesis.

Effects of cryosolvents and D2O/H2O substitution on the reaction centres (RCs) isolated from photosynthetic bacteria were studied with respect to the role of intra-protein hydrogen bonds in the primary photosynthetic electron transfer. As a result of such treatment of RCs, the charge separation rate between the photoactive bacteriochlorophyll (P2 dimer) and bacteriopheophytin and the rate of electron transfer to the primary quinone slowed down. The energy migration rate from bacteriopheophytin (BPheM), inactive in electron transport, to P2 decreased as well. Although cryosolvents can shift the redox potential of the photoactive pigment, there is no direct correlation between the P2 potential and the effects of these modifying agents on the photosynthetic process in RCs occurring with participation of P2. The removal of H subunit from the pigment-protein complex results in the pronounced weakening of the dimethyl sulfoxide modifying effects on the RC hydrogen bonds. The role of structural and dynamic state in the functioning of the photosynthetic bacterial RCs is analyzed. Relaxation processes in purple bacteria RCs accompanying the primary picosecond steps of energy transformation proceed with the participation of small proton-containing molecular groups in the immediate surroundings of electron transfer carriers. In this paper, we present results concerning mechanisms of primary photosynthetic steps, which were initiated by A. A. Krasnovsky and have been studied for several years at the Department of Biophysics. This paper is dedicated to the memory of our teacher Prof. A. A. Krasnovsky.     

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs

Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone Q_A, non-heme Fe²⁺ ion and neighboring residues_. The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the Q_A state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [Q_A-His^M219] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P⁺/Q_A⁻ recombination rate temperature dependence.     

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs

Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone Q(A), non-heme Fe(2+) ion and neighboring residues(.) The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the Q(A) state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [Q(A)-His(M219)] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P(+)/Q(A)(-) recombination rate temperature dependence.     

Effect of energy status of hydrogen bond protons on the rate of electron transfer in photosynthetic reaction centers

We present here a theoretical interpretation of the temperature dependence of the rate of dark recombination which takes place in Rhodobacter sphaeroides reaction centers between a primary quinone (Q(A)) and a bacteriochlorophyll dimer. Taking the energy of interaction between hydrogen bond protons and an excessive electron into account, we described qualitative by this nonmonotonous dependence. We considered a molecular model of the primary quinone from Rb. sphaeroides reaction centers. In addition to the primary quinone, the model includes two reaction center fragments that form hydrogen bonds with Q(A). One of these fragments is His(M219), and the other is the peptide [Asn(M259) - Ala(M260)]. We used the two-center approach with regard for electron-phonon interaction in order to calculate the characteristic time of electron tunneling during the recombination reaction. The energy of the phonon emitted/absorbed during the electron tunneling is determined by a relative shift of the donor and the acceptor energy levels, the detuning of levels. The value of level detuning was shown to be temperature dependent in a nonmonotonous manner in the case of hydrogen bonds with double-well potential energy surface. The characteristic time (or the reaction rate) depends on temperature parametrically. The dependence is nonmonotonous and is in qualitative agreement with the experimental one.     

Evidence for hydrogen bond formation to the PsaB chlorophyll of P700 in photosystem I mutants of Synechocystis sp. PCC 6803

P700, the primary electron donor of photosystem I, is an asymmetric dimer made of one molecule of chlorophyll a' (P(A)) and one of chlorophyll a (P(B)) that are bound to the homologous PsaA and PsaB polypeptides. While the carbonyl groups of P(A) are involved in hydrogen-bonding interactions with several surrounding amino acid side chains and a water molecule, P(B) does not engage hydrogen bonds with the protein. Notably, the residue Thr A739 is donating a strong hydrogen bond to the 9-keto C=O group of P(A) and the homologous residue Tyr B718 is free from interaction with P(B). Light-induced FTIR difference spectroscopy of the photooxidation of P700 has been combined with a site-directed mutagenesis attempt to introduce hydrogen bonds to the carbonyl groups of P(B) in Synechocystis sp. PCC 6803. The FTIR study of the Y(B718)T mutant provides evidence that the 9-keto C=O group of P(B) and P(B)(+) engages a relatively strong hydrogen-bonding interaction with the surroundings in a significant fraction (40 +/-10%) of the reaction centers. Additional mutations on the two PsaB residues homologous to those involved in the main interactions between the PsaA polypeptide and the 10a-carbomethoxy groups of P(A) affect only marginally the vibrational frequency of the 10a-ester C=O group of P(B). The FTIR data on single, double, and triple mutants at these PsaB sites indicate a plasticity of the interactions of the carbonyl groups of P(B) with the surrounding protein. However, these mutations do not perturb the hydrogen-bonding interactions assumed by the 9-keto and 10a-ester C=O groups of P(A) and P(A)(+) with the protein and have only a limited effect on the relative charge distribution between P(A)(+) and P(B)(+).     

Pigment-protein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis

An X-ray structure analysis of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis provides structural details of the pigment-binding sites. The photosynthetic pigments are found in rather hydrophobic environments provided by the subunits L and M. In addition to apolar interactions, the bacteriochlorophylls of the primary electron donor (`special pair') and the bacteriopheophytins, but not the accessory bacteriochlorophylls, form hydrogen bonds with amino acid side chains of these protein subunits. The two branches of pigments which originate at the primary electron donor, and which mark possible electron pathways across the photosynthetic membrane, are in different environments and show different hydrogen bonding with the protein: this may help to understand why only one branch of pigments is active in the light-driven electron transfer. The primary electron acceptor, a menaquinone (Q_A), is in a pocket formed by the M subunit and interacts with it by hydrophobic contacts and hydrogen bonds. Competitive inhibitors of the secondary quinone Q_B (o-phenanthroline, the herbicide terbutryn) are bound into a pocket provided by the L subunit. Apart from numerous van der Waals interactions they also form hydrogen bonds to the protein.     

The functions of tryptophan residues in membrane proteins.

Membrane proteins have a significantly higher Trp content than do soluble proteins. This is especially true for the M and L subunits of the photosynthetic reaction center from purple bacteria. The Trp residues are not uniformly distributed through the membrane but are concentrated at the periplasmic side of the complex. In addition, Trp residues are not randomly aligned. Within the protein subunits, many form hydrogen bonds with carbonyl oxygens of the main chain, thereby stabilizing the protein. On the surface of the molecule, they are correctly positioned to form hydrogen bonds with the lipid head groups while their hydrophobic rings are immersed in the lipid part of the bilayer. These observations suggest that Trp residues are involved in the translocation of protein through the membrane and that following translocation, Trp residues serve as anchors on the periplasmic side of the membrane.     

Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex

We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc₁ complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (b_H heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b_H heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the b_H heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.     

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.Abbreviations Bchl bacteriochlorophyll - Bpheo bacteriopheophytin - D electron donor to P⁺ - P bacteriochlorophyll dimer - Q quinone acceptor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ₆ ubiquinone-30     

Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: I. Identification of the ENDOR lines associated with the hydrogen bonds to the primary quinone QA*-

Hydrogen bonds are important in determining the structure and function of biomolecules. Of particular interest are hydrogen bonds to quinones, which play an important role in the bioenergetics of respiration and photosynthesis. In this work we investigated the hydrogen bonds to the two carbonyl oxygens of the semiquinone QA*- in the well-characterized reaction center from the photosynthetic bacterium Rhodobacter sphaeroides R-26. We used electron paramagnetic resonance and electron nuclear double resonance techniques at 35 GHz at a temperature of 80 K. The goal of this study was to identify and assign sets of 1H-ENDOR lines to protons hydrogen bonded to each of the two oxygens. This was accomplished by preferentially exchanging the hydrogen bond on one of the oxygens with deuterium while concomitantly monitoring the changes in the amplitudes of the 1H-ENDOR lines. The preferential deuteration of one of the oxygens was made possible by the different 1H --> 2H exchange times of the protons bonded to the two oxygens. The assignment of the 1H-ENDOR lines sets the stage for the determination of the geometries of the H-bonds by a detailed field selection ENDOR study to be presented in a future article.     

Strong effects of an individual water molecule on the rate of light-driven charge separation in the Rhodobacter sphaeroides reaction center

The role of a water molecule (water A) located between the primary electron donor (P) and first electron acceptor bacteriochlorophyll (B(A)) in the purple bacterial reaction center was investigated by mutation of glycine M203 to leucine (GM203L). The x-ray crystal structure of the GM203L reaction center shows that the new leucine residue packs in such a way that water A is sterically excluded from the complex, but the structure of the protein-cofactor system around the mutation site is largely undisturbed. The results of absorbance and resonance Raman spectroscopy were consistent with either the removal of a hydrogen bond interaction between water A and the keto carbonyl group of B(A) or a change in the local electrostatic environment of this carbonyl group. Similarities in the spectroscopic properties and x-ray crystal structures of reaction centers with leucine and aspartic acid mutations at the M203 position suggested that the effects of a glycine to aspartic acid substitution at the M203 position can also be explained by steric exclusion of water A. In the GM203L mutant, loss of water A was accompanied by an approximately 8-fold slowing of the rate of decay of the primary donor excited state, indicating that the presence of water A is important for optimization of the rate of primary electron transfer. Possible functions of this water molecule are discussed, including a switching role in which the redox potential of the B(A) acceptor is rapidly modulated in response to oxidation of the primary electron donor.     

Study of effect of molecular mobility in chromatophore membranes of the bacterium <Emphasis Type="Italic">E. shaposhnikovii</Emphasis> on processes of photoinduced electron transport using the NMR-Spin-Echo method with isotope substitution and dehydration

The effect of dehydration and ²H₂O/H₂O isotope substitution on electron transport reactions and relaxation of proton-containing groups was studied in chromatophore membranes of Ectothiorhodospira shaposhnikovii. During dehydration (including isotope substitution of hydrate water) of preliminarily dehydrated isolated photosynthetic membranes there was a partial correlation between hydration intervals within which activation of electron transport from high-potential cytochrome c to photoactive bacteriochlorophyll dimer P890 of photosynthetic reaction center and variation of spin-lattice and spin-spin proton relaxation time was observed. Partial correlation between hydration intervals can be considered as evidence of correlation between mobility of non-water proton-containing groups with proton relaxation frequency ∼10⁸ sec⁻¹ with efficiency of electron transfer at the donor side of the chain.     

Conformation-Determining role for the N-acetyl group in the O-glycosidic linkage, α-GalNAc-Thr

An ¹H-nmr study of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-galactopyranose (AcGalNAc) glycosylated Thr-containing tripeptides in Me₂SO-d₆ solution reveals two mutually exclusive intramolecular hydrogen bonds. In Z-Thr(AcGalNAc)-Ala-Ala-OMe, there is an intramolecular hydrogen bond between the Thr amide proton and the sugar N-acetyl carbonyl oxygen. The strength of this hydrogen bond will be dependent on the amino acid residues on the Thr C terminal side to some undetermined distance. In Ac-Thr(AcGalNAc)-Ala-Ala-OMe, a different intramolecular hydrogen bond between the sugar N-acetyl amide proton and the Thr carbonyl oxygen exists. The choice of hydrogen bonds seems dependent on the bulkiness of the residues on the Thr N terminal side. The consequence of such strong hydrogen bonds is a clearly defined orientation of the sugar moiety with respect to the peptide backbone. In the former, the plane of the sugar pyranose ring is roughly oriented perpendicularly to the peptide backbone. The latter orientation is where the plane of the sugar ring is roughly in line with the peptide backbone. In both orientations, the sugar moiety can increase the shielding of the neighboring amino acid residues from the solvent. The idea that the amino acid residues near the glycosylated Thr influence orientation of the sugar moiety with respect to the peptide backbone and in turn possibly hinder peptide backbone flexibility has interesting implications in the conformational as well as the biological role of O-glycoproteins.     

Dissipation in bioenergetic electron transfer chains

This paper examines the processes by which wasteful dissipation of free energy may occur in bioenergetic electron transfer chains. Frictionless transfer requires high rate constants in order to achieve a quasi-equilibrium steady-state. Previous results concerning the maximum power available from a photochemical source are recalled. The energetic performance of the bacterial reaction center is discussed, characterizing the processes that decrease either the quantum yield (recombination and obstruction) or the chemical potential (friction and non-equilibrated mechanisms). Considering the whole chain, diffusive carriers are potentially weaker links, due to kinetic limitation and short-circuiting reactions. It is suggested that the evolutionary trend has been to limit their number by lumping them into tightly bound protein complexes or, in a more flexible way, into labile supercomplexes.Abbreviations Cyt cytochrome - F Faraday - H primary acceptor in the bacterial reaction center (bacteriopheophytin) - k _B Boltzmann's constant - P primary photochemical donor (special bacteriochlorophyll pair) - RC reaction center - Q_A, Q_B primary, secondary quinone acceptor     

A possible biological role of the electron transfer between tyrosine and tryptophan. Gating of ion channels.

Experiments have demonstrated that four tryptophan residues are located near the tetrodotoxin binding site in Na+ channels, and that conserved tyrosine and tryptophan residues are located in the pore-forming region of voltage-sensitive K+ channels. This paper proposes an activation mechanism involving electron transfer between these residues. The K+ channel may be closed by four tyrosine residues forming hydrogen bonds with each other. After electron transfer, these hydrogen bonds will be broken, thereby opening the channel. The Na+ channel could be activated by a similar mechanism. This idea can be tested directly by observing tyrosine or tryptophan radicals when the channels are in the open state.