Electrostatic interactions between charged amino acid residues and the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides.

The extent of electrostatic contributions from the protein environment was assessed by the introduction of ionizable residues near the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides. Two mutations at symmetry-related sites, M199 Asn to Asp and L170 Asn to Asp, resulted in a 48 and 44 mV lowering of the midpoint potential, respectively, compared to the wild type at pH 8, while a 75 mV decrease in the midpoint potential was observed for the mutation L168 His to Glu. The decrease relative to wild type was found to be approximately additive, up to 147 mV, for various combinations of the mutations. As the pH was lowered from 9.5 to 6.0, the relative decrease in the midpoint potential became smaller for each of these three mutations. Titration of the pH dependence of the change in midpoint potential of the M199 Asn to Asp mutant compared to wild type yielded a pK(a) value of 7.9 and a change in midpoint potential from low to high pH of 59 mV. The major effect of the mutation on the midpoint potential of the dimer is interpreted as stemming from a negative charge on the residue. An average dielectric constant of approximately 20 was estimated for the local protein environment, consistent with a relatively hydrophobic environment for residue M199. The rate of charge recombination between the primary quinone acceptor and the bacteriochlorophyll dimer decreased in the M199 Asn to Asp mutant at high pH, reflecting the decrease in midpoint potential.     

Effects of ionizable residues on the absorption spectrum and initial electron-transfer kinetics in the photosynthetic reaction center of Rhodobacter sphaeroides

Effects of ionizable amino acids on spectroscopic properties and electron-transfer kinetics in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are investigated by site-directed mutations designed to alter the electrostatic environment of the bacteriochlorophyll dimer that serves as the photochemical electron donor (P). Arginine residues at homologous positions in the L and M subunits (L135 and M164) are changed independently: Arg L135 is replaced by Lys, Leu, Glu, and Gln and Arg M164 by Leu and Glu. Asp L155 also is mutated to Asn, Tyr L164 to Phe, and Cys L247 to Lys and Asp. The mutations at L155, L164, and M164 have little effect on the absorption spectrum, whereas those at L135 and L247 shift the long-wavelength absorption band of P to higher energies. Fits to the ground-state absorption and hole-burned spectra indicate that the blue shift and increased width of the absorption band in the L135 mutants are due partly to changes in the distribution of energies for the zero-phonon absorption line and partly to stronger electron-phonon coupling. The initial electron-transfer kinetics are not changed significantly in most of the mutants, but the time constant increases from 3.0 +/- 0.2 in wild-type RCs to 4.7 +/- 0.2 in C(L247)D and 7.0 +/- 0.3 ps in C(L247)K. The effects of the mutations on the solvation free energies of the product of the initial electron-transfer reaction (P(+)) and the charge-transfer states that contribute to the absorption spectrum ( and ) were calculated by using a distance-dependent electrostatic screening factor. The results are qualitatively in accord with the view that electrostatic interactions of the bacteriochlorophylls with ionized residues of the protein are strongly screened and make only minor contributions to the energetics and dynamics of charge separation. However, the slowing of electron transfer in the Cys L247 mutants and the blue shift of the spectrum in some of the Arg L135 and Cys L247 mutants cannot be explained consistently by electrostatic interactions of the mutated residues with P and B(L); we ascribe these effects tentatively to structural changes caused by the mutations.     

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.     

Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides

The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues. One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well. These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV. The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation. These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe. Radicals were produced in the mutants by using light to initiate electron transfer. The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra. The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164. The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.     

The structure of the heterodimer reaction center from Rhodobacter sphaeroides at 2.55 å resolution

Crystals have been obtained of reaction centers of the heterodimer mutant that has significantly different properties than wild type due to the primary donor being formed from both a bacteriochlorophyll and bacteriopheophytin rather than two bacteriochlorophylls as found for wild type. The crystals belong to the trigonal space group P3(1)21 and the structure has been refined to a resolution limit of 2.55 A with an R factor of 19.0%. The electron density maps confirm that a primary donor does indeed contain a bacteriopheophytin due to the His to Leu substitution at M202 that coordinates the corresponding bacteriochlorophyll in wild-type. Other structural changes compared to wild type are relatively minor with the relative orientation and positioning of the two tetrapyrroles forming the primary donor being unchanged within the error. Compared to wild type, the only significant alterations are small shifts of residues M196 to M206, a rotation of the side chain of Ile M206, and the loss of a bound water molecule that in wild-type is hydrogen-bonded to both His M202 and the bacteriochlorophyll monomer on the active branch. Since hydrogen-bonding interactions strongly influence the energies of tetrapyrroles, the loss of the water molecule should result in changes in the energies of the bacteriochlorophyll monomer that contributes to the observed functional differences with wild-type.     

Tuning of the optical and electrochemical properties of the primary donor bacteriochlorophylls in the reaction centre from Rhodobacter sphaeroides: spectroscopy and structure

A series of mutations have been introduced at residue 168 of the L-subunit of the reaction centre from Rhodobacter sphaeroides. In the wild-type reaction centre, residue His L168 donates a strong hydrogen bond to the acetyl carbonyl group of one of the pair of bacteriochlorophylls (BChl) that constitutes the primary donor of electrons. Mutation of His L168 to Phe or Leu causes a large decrease in the mid-point redox potential of the primary electron donor, consistent with removal of this strong hydrogen bond. Mutations to Lys, Asp and Arg cause smaller decreases in redox potential, indicative of the presence of weak hydrogen bond and/or an electrostatic effect of the polar residue. A spectroscopic analysis of the mutant complexes suggests that replacement of the wild-type His residue causes a decrease in the strength of the coupling between the two primary donor bacteriochlorophylls. The X-ray crystal structure of the mutant in which His L168 has been replaced by Phe (HL168F) was determined to a resolution of 2.5 A, and the structural model of the HL168F mutant was compared with that of the wild-type complex. The mutation causes a shift in the position of the primary donor bacteriochlorophyll that is adjacent to residue L168, and also affects the conformation of the acetyl carbonyl group of this bacteriochlorophyll. This conformational change constitutes an approximately 27 degrees through-plane rotation, rather than the large into-plane rotation that has been widely discussed in the context of the HL168F mutation. The possible structural basis of the altered spectroscopic properties of the HL168F mutant reaction centre is discussed, as is the relevance of the X-ray crystal structure of the HL168F mutant to the possible structures of the remaining mutant complexes.     

Manipulating the direction of electron transfer in the bacterial reaction center by swapping Phe for Tyr near BChl(M) (L181) and Tyr for Phe near BChl(L) (M208)

We have investigated the primary charge separation processes in Rb. capsulatus reaction centers (RCs) bearing the mutations Phe(L181) --> Tyr, Tyr(M208) --> Phe, and Leu(M212) --> His. In the YFH mutant, decay of the excited primary electron donor P occurs with an 11 +/- 2 ps time constant and is trifurcated to give (1) internal conversion to the ground state ( approximately 10% yield), (2) charge separation to the L side of the RC ( approximately 60% yield), and (3) electron transfer to the M-side bacteriopheophytin BPh(M) ( approximately 30% yield). These results relate previous work in which the ionizable residues Lys (at L178) and Asp (at M201) have been used to facilitate charge separation to the M side of the RC, and the widely studied L181 and M208 mutants. One conclusion that comes from this work is that the Tyr (M208) --> Phe and Gly(M201) --> Asp mutations near the L-side bacteriochlorophyll (BChl(L)) raise the free energy of P(+)BChl(L)(-) by comparable amounts. The results also suggest that the free energy of P(+)BChl(M)(-) is lowered more substantially by a Tyr at L181 than a Lys at L178. The results on the YFH mutant further demonstrate that the free energy differences between the L- and M-side charge-separated states play a significant role in the directionality of charge separation in the wild-type RC, and place limits on the contributing role of differential electronic matrix elements on the two sides of the RC.     

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.     

A new infrared electronic transition of the oxidized primary electron donor in bacterial reaction centers: a way to assess resonance interactions between the bacteriochlorophylls.

The primary electron donor in the reaction center of purple photosynthetic bacteria consists of a pair of bacteriochlorophylls (PL and PM). The oxidized dimer (P+) is expected to have an absorption band in the mid-IR, whose energy and dipole strength depend in part on the resonance interactions between the two bacteriochlorophylls. A broad absorption band with the predicted properties was found in a previously unexplored region of the spectrum, centered near 2600 cm-1 in reaction centers of Rhodobacter sphaeroides and several other species of bacteria that contain bacteriochlorophyll a, and near 2750 cm-1 in Rhodopseudomonas viridis. The band is not seen in the absorption spectrum of the monomeric bacteriochlorophyll cation in solution, and it is missing or much diminished in the reaction centers of bacterial mutants that have a bacteriopheophytin in place of either PL or PM. With the aid of a relatively simple quantum mechanical model, the measured transition energy and dipole strength of the band can be used to solve for the resonance interaction matrix element that causes an electron to move back and forth between PL and PM, and also for the energy difference between states in which a positive charge is localized on either PL or PM. (The absorption band can be viewed as representing a transition between supermolecular eigenstates that are obtained by mixing these basis states.) The values of the matrix element obtained in this way agree reasonably well with values calculated by using semiempirical atomic resonance integrals and the reaction center crystal structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tuning of the redox potential of the primary electron donor in reaction centres of purple bacteria: effects of amino acid polarity and position

Mutation of residues His L168 and Phe M197 in the reaction centre from Rhodobacter sphaeroides has an unusually strong effect on the mid-point redox potential (E(m)) of the pair of bacteriochlorophylls that form the primary donor of electrons, tuning E(m) over a range of nearly 250 mV. This effect is correlated to the accompanying change in the permanent dipole of the L168 or M197 residue, suggesting it is mediated by changes in charge-dipole interactions. Comparisons with mutations made at a variety of other positions show that this correlation is particular to this residue pair, perhaps reflecting their proximity to the ring I regions of the dimer bacteriochlorophylls that form the overlap region between these molecules.     

Investigation into the source of electron transfer asymmetry in bacterial reaction centers

We have investigated the primary photochemistry of two symmetry-related mutants of Rhodobacter sphaeroides in which the histidine residues associated with the central Mg2+ ions of the two bacteriochlorophylls of the dimeric primary electron donor (His-L173 and His-M202) have been changed to leucine, affording bacteriochlorophyll (BChl)/bacteriopheophytin (BPh) heterodimers. Reaction centers (RCs) from the two mutants, (L)H173L and (M)H202L, have remarkably similar spectral and kinetic properties, although they are quite different from those of wild-type RCs. In both mutants, as in wild-type RCs, electron transfer to BPhL and not to BPhM is observed. These results suggest that asymmetry in the charge distribution of the excited BChl dimer (P*) in wild-type RCs (due to differing contributions of the two opposing intradimer charge-transfer states) contributes only modestly to the directionality of electron transfer. The results also suggest that differential orbital overlap of the two BChls of P with the chromophores on the L and M polypeptides does not contribute substantially to preferential electron transfer to BPhL.     

Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: High-field EPR study

High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D⁺ couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone-iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.     

Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand.     

Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids

To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.     

Manipulating monomer-dimer equilibrium of bovine Beta -lactoglobulin by amino acid substitution

Bovine beta-lactoglobulin, a major protein in cow's milk composed of nine beta-strands (betaA-betaI) and one alpha-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0. It is assumed that the intermolecular beta-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation. Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation. Various I-strand mutants showed decreases in K(a), suggesting that the intermolecular beta-sheet is essential for dimer formation. By substituting either Asp(33) or Arg(40) on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in K(a) was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg(40) on one monomer and Asp(33) on the other monomer in the wild-type dimer. However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg(33) and Asp(40) of different molecules. These results suggested that protein-protein interactions of bovine beta-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.     

Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants.

To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.     

Electrostatic interactions in an integral membrane protein

The effects of charge-charge interactions on the midpoint reduction potential (E(m)()) of the primary electron donor (P) in the photosynthetic reaction center of Rhodobacter sphaeroides were investigated by introducing mutations of ionizable amino acids at selected sites. The mutations were designed to alter the electrostatic environment of P, a bacteriochlorophyll dimer, without greatly affecting its structure or molecular orbitals. Two arginine residues at homologous positions in the L and M subunits [residues (L135) and (M164)], Asp (L155), Tyr (L164), and Cys (L247) were changed independently. Arginine (L135) was replaced by Lys, Leu, Gln, or Glu; Arg (M164), by Leu or Glu; Asp (L155), by Asn; Tyr (L164), by Phe; and Cys (L247), by Lys or Asp. The R(L135)E/C(L247)K double mutant also was made. The shift in the E(m)() of P/P(+) was measured in each mutant and was compared with the effect predicted by electrostatics calculations using several different computational approaches. A simple distance-dependent dielectric screening factor reproduced the effects remarkably well. By contrast, microscopic methods that considered the reaction field in the protein and solvent but did not include explicit counterions overestimated the changes in the E(m)() considerably. Including counterions for the charged residues reduced the calculated effects of the mutations in molecular dynamics calculations. The results show that electrostatic interactions of P with ionizable amino acid residues are strongly screened, and suggest that counterions make major contributions to this screening. The screening also could reflect penetration of water or other relaxations not taken into account because of incomplete sampling of configurational space.     

Structural consequences of the replacement of glycine M203 with aspartic acid in the reaction center from Rhodobacter sphaeroides

Reaction centers with the double mutation Phe M197 to Arg and Gly M203 to Asp (FM197R/GM203D) have been crystallized from an antenna-deficient strain of Rhodobacter sphaeroides, and the structure has been determined at 2.7 A resolution. Unlike in reaction centers with a single FM197R mutation, the Arg M197 residue in the FM197R/GM203D reaction center adopts a position similar to that of the native Phe residue in the wild-type reaction center. Asp M203 is packed in such a way that the gamma-carboxy group interacts with the backbone carbonyl of Arg M197. The Asp M203 residue takes up part of the volume that is occupied in the wild-type reaction center by a water molecule. This water has been proposed to form a hydrogen bond interaction with the 9-keto carbonyl group of the active branch accessory bacteriochlorophyll, particularly when the primary donor bacteriochlorophylls are oxidized. The GM203D mutation therefore appears to remove the possibility of this hydrogen bond interaction by exclusion of this water molecule, as well as altering the local dielectric environment of the 9-keto carbonyl group. We examine whether the observed structural changes can provide new or alternative explanations for the absorbance and electron-transfer properties of reaction centers with the FM197R and GM203D mutations.     

Crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin from Thiobacillus ferrooxidans: insights into the structural relationship with the cupredoxins and the multi copper proteins

The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65 A resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant ( approximately 800 mV) is elevated compared to that of the native protein ( approximately 670 mV at pH 3.2) while that of the Ser86Asp mutant ( approximately 623 mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.     

EPR, ENDOR, and Special TRIPLE measurements of P•+ in wild type and modified reaction centers from Rb. sphaeroides

The influence of the protein environment on the primary electron donor, P, a bacteriochlorophyll a dimer, of reaction centers from Rhodobacter sphaeroides, has been investigated using electron paramagnetic resonance and electron nuclear double resonance spectroscopy. These techniques were used to probe the effects on P that are due to alteration of three amino acid residues, His L168, Asn L170, and Asn M199. The introduction of Glu at L168, Asp at L170, or Asp at M199 changes the oxidation/reduction midpoint potential of P in a pH-dependent manner (Williams et al. (2001) Biochemistry 40, 15403-15407). For the double mutant His L168 to Glu and Asn at L170 to Asp, excitation results in electron transfer along the A-side branch of cofactors at pH 7.2, but at pH 9.5, a long-lived state involving B-side cofactors is produced (Haffa et al. (2004) J Phys Chem B 108, 4-7). Using electron paramagnetic resonance spectroscopy, the mutants with alterations of each of the three individual residues and a double mutant, with changes at L168 and L170, were found to have increased linewidths of 10.1-11.0 G compared to the linewidth of 9.6 G for wild type. The Special TRIPLE spectra were pH dependent, and at pH 8, the introduction of aspartate at L170 increased the spin density ratio, rho (L)/rho (M), to 6.1 while an aspartate at the symmetry related position, M199, decreased the ratio to 0.7 compared to the value of 2.1 for wild type. These results indicate that the energy of the two halves of P changes by about 100 meV due to the mutations and are consistent with the interpretation that electrostatic interactions involving these amino acid residues contribute to the switch in pathway of electron transfer.