Biomedical subjects

G Feher

Publications and source records attributed to G Feher.

At least 19 recordsLinked to original sources

PubMed1998

Conformational gating of the electron transfer reaction QA-.QB --> QAQB-. in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay.

The mechanism of the electron transfer reaction, QA-.QB --> QAQB-., was studied in isolated reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides by replacing the native Q10 in the QA binding site with quinones having different redox potentials. These substitutions are expected to change the intrinsic electron transfer rate by changing the redox free energy (i.e., driving force) for electron transfer without affecting other events that may be associated with the electron transfer (e.g., protein dynamics or protonation). The electron transfer from QA-. to QB was measured by three independent methods: a functional assay involving cytochrome c2 to measure the rate of QA-. oxidation, optical kinetic spectroscopy to measure changes in semiquinone absorption, and kinetic near-IR spectroscopy to measure electrochromic shifts that occur in response to electron transfer. The results show that the rate of the observed electron transfer from QA-. to QB does not change as the redox free energy for electron transfer is varied over a range of 150 meV. The strong temperature dependence of the observed rate rules out the possibility that the reaction is activationless. We conclude, therefore, that the independence of the observed rate on the driving force for electron transfer is due to conformational gating, that is, the rate limiting step is a conformational change required before electron transfer. This change is proposed to be the movement, controlled kinetically either by protein dynamics or intermolecular interactions, of QB by approximately 5 A as observed in the x-ray studies of Stowell et al. [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E. & Feher, G. (1997) Science 276, 812-816].

Binding Sites↗

PubMed1997

Proton and electron transfer to the secondary quinone (QB) in bacterial reaction centers: the effect of changing the electrostatics in the vicinity of QB by interchanging asp and glu at the L212 and L213 sites.

The bacterial reaction center (RC) plays a central role in photosynthetic energy conversion by facilitating the light induced double reduction and protonation of a bound quinone molecule, QB. Two carboxylic acid residues, Asp-L213 and Glu-L212, located near QB, were previously shown to be important for proton transfer to QB. In this work, the ability of Glu to substitute for Asp at L213 and Asp to substitute for Glu at L212 was tested by site-directed mutagenesis. Both single mutants and a double mutant in which Asp and Glu were exchanged between the two sites were constructed. The electron transfer rate constants kBD (D+QAQB- --> DQAQB), and kAB(2) (DQA-QB- + H+ --> DQA(QBH)-), that are known to be sensitive to the energy of the QB- state, were found to be altered by Asp/Glu substitutions. Both rates were fastest ( approximately 10-fold) in RCs with Asp at both sites, slowest with Glu at both sites ( approximately 50-fold) and relatively unchanged by the caboxylic acid exchange. These changes could be explained if Asp was predominantly ionized and Glu was predominantly protonated at both sites (pH 7.5). The charge recombination kBD suggests an observed approximately 5 pKa unit difference of Glu over Asp. Modeling of kBD by strong electrostatic interactions ( approximately 3-4 pKa units) among negatively charged acids and QB- indicated a lower intrinsic pKa for Asp compared to Glu at either site of approximately 2-3 units. The mechanism of the kAB(2) reaction was determined to be the same in all mutant RCs as for native RCs. A quantitative explanation of the effect of the electrostatic environment on kAB(2) was obtained using the two-step model proposed for native RCs [Graige, M. S., Paddock, M. L., Bruce, J. M., Feher, G., & Okamura, M. Y. (1996) J. Am. Chem. Soc. 118, 9005-9016] which involves fast protonation of the semiquinone followed by rate-limiting electron transfer. Using simple models for the quinone/quinol conversion rate, it is shown that the optimal electrostatic potential for the QB site is close to that found in native RCs.

Aspartic Acid↗

PubMed1997

Light-induced electrogenic events associated with proton uptake upon forming QB- in bacterial wild-type and mutant reaction centers.

Light-induced voltage changes (electrogenic events) were measured in wild-type and site-directed mutants of reaction centers (RCs) from Rhodobacter sphaeroides oriented in a lipid monolayer adsorbed to a Teflon film. A rapid increase in voltage associated with charge separation was followed by a slower increase attributed to proton transfer from solution to protonatable amino-acid residues in the vicinity of the QB site. In native reaction centers the proton-transfer voltage had a pH-dependent amplitude with two peaks at pH 4.5 and pH 9.7, respectively. In the Glu-L212-->Gln RCs the high-pH peak was absent, whereas in the Asp-L213-->Asn RCs the low-pH peak was absent and the high-pH peak was shifted to lower pH by about 1.3 pH units. The amplitudes of the electrogenic phases as a function of pH follow approximately the measured proton uptake from solution (P.H. McPherson, M.Y. Okamura, G. Feher, Biochim. Biophys. Acta, vol. 934, 1988, pp. 348-368) and are ascribed to proton transfer to amino acid residues upon QB- formation. The peak around pH 9.7 is ascribed to proton uptake predominantly by Glu-L212 and the peak around pH 4.5 to proton uptake predominantly by Asp-L213 or a residue strongly interacting with Asp-L213.

Hydrogen-Ion Concentration↗

PubMed1997

Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer.

High resolution x-ray diffraction data from crystals of the Rhodobacter sphaeroides photosynthetic reaction center (RC) have been collected at cryogenic temperature in the dark and under illumination, and the structures were refined at 2.2 and 2.6 angstrom resolution, respectively. In the charge-separated D+QAQB- state (where D is the primary electron donor (a bacteriochlorophyll dimer), and QA and QB are the primary and secondary quinone acceptors, respectively), QB- is located approximately 5 angstroms from the QB position in the charge-neutral (DQAQB) state, and has undergone a 180 degrees propeller twist around the isoprene chain. A model based on the difference between the two structures is proposed to explain the observed kinetics of electron transfer from QA-QB to QAQB- and the relative binding affinities of the different ubiquinone species in the QB pocket. In addition, several water channels (putative proton pathways) leading from the QB pocket to the surface of the RC were delineated, one of which leads directly to the membrane surface.

Binding Sites↗

PubMed1996

Co-crystallization and characterization of the photosynthetic reaction center-cytochrome c2 complex from Rhodobacter sphaeroides.

The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c2 (cyt c2), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c2 was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 angstroms. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 angstroms. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 angstroms and c = 254.8 angstroms. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c2 by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c2 was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c2, and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c2 amino acid residues. The RC-cyt c2 structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c2 reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 micros in the co-crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC-cyt c2 complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c2 on the RC. The cyt c2 position with the lowest electrostatic energy is very similar to that of the cyt c2 in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M184 and L155) on the periplasmic surface of the RC. Cyt c2 binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of th RC-cyt c2 complex.

Binding Sites↗

PubMed1996

Protein crystallization.

Crystallization is necessary to obtain the three-dimensional structure of proteins and nucleic acids; it often represents the bottleneck in structure determination. Our understanding of crystallization mechanisms is still incomplete. In this review, we emphasize fundamental aspects of the crystallization process. Protein-protein contacts in crystals are complex, involving a delicate balance of specific and nonspecific interactions. Depending on solution conditions, these interactions can lead to nucleation of crystals or to amorphous aggregation; this stage of crystallization has been successfully studied by light scattering. Post-nucleation crystal growth may proceed by mechanisms involving crystal defects or two-dimensional nucleation, as observed by atomic force and interference microscopy. Cessation of growth has been observed but remains incompletely understood. Impurities may play important roles during all stages of crystallization. Phase diagrams can guide optimization of conditions for nucleation and subsequent crystal growth; a theoretical understanding relating these to the intermolecular interactions is beginning to develop.

Animals↗

PubMed1995

Pathway of proton transfer in bacterial reaction centers: further investigations on the role of Ser-L223 studied by site-directed mutagenesis.

The role of Ser-L223 in proton transfer to reduced QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Ser with residues having different proton donor properties, e.g., the aliphatic residues Ala and Gly, the hydroxyl residue Thr, the amide residue Asn, the sulhydryl residue Cys, the imidazole residue His, and the carboxylic acid residue Asp. Compared to native reaction centers, RCs with Ala or Asn at L223 had greatly reduced (approximately 300-fold) proton-coupled electron transfer rates, kAB(2), associated with the second electron reduction of QB (QA(-)QB(-) + H+ --> QAQBH-). In contrast, RCs containing Thr, Asp, or Gly at L223 retained fast proton-coupled electron transfer rates. RCs with His or Cys at L223 did not bind the secondary quinone QB. These results show that kAB(2) is larger when a good proton transfer group, e.g., a hydroxyl residue (Ser, Thr) or a carboxylic acid (Asp), occupies the L223 site, supporting the proposal that Ser-L223 is a component of a proton transfer chain [Paddock, M. L., McPherson, P. H., Feher, G., & Okamura, M. Y. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6803--6807]. The surprising result that kAB(2) is not significantly reduced in RCs with Gly at L223 suggests that a water molecule functionally replaces the missing Ser hydroxyl group in the mutant RCs. The importance of Ser-L223 in internal proton transfer reactions within the RC is discussed.

Base Sequence↗

PubMed1995

Electronic structure of Q-A in reaction centers from Rhodobacter sphaeroides. I. Electron paramagnetic resonance in single crystals.

The magnitude and orientation of the electronic g-tensor of the primary electron acceptor quinone radical anion, Q-A, has been determined in single crystals of zinc-substituted reaction centers of Rhodobacter sphaeroides R-26 at 275 K and at 80 K. To obtain high spectral resolution, EPR experiments were performed at 35 GHz and the native ubiquinone-10 (UQ10) in the reaction center was replaced by fully deuterated UQ10. The principal values and the direction cosines of the g-tensor axes with respect to the crystal axes a, b, c were determined. Freezing of the single crystals resulted in only minor changes in magnitude and orientation of the g-tensor. The orientation of Q-A as determined by the g-tensor axes deviates only by a few degrees (< or = 8 degrees) from the orientation of the neutral QA obtained from an average of four different x-ray structures of Rb. sphaeroides reaction centers. This deviation lies within the accuracy of the x-ray structure determinations. The g-tensor values measured in single crystals agree well with those in frozen solutions. Variations in g-values between Q-A, Q-B, and UQ10 radical ion in frozen solutions were observed and attributed to different environments.

Anions↗

PubMed1995

Electrostatic calculations of amino acid titration and electron transfer, Q-AQB-->QAQ-B, in the reaction center.

The titration of amino acids and the energetics of electron transfer from the primary electron acceptor (QA) to the secondary electron acceptor (QB) in the photosynthetic reaction center of Rhodobacter sphaeroides are calculated using a continuum electrostatic model. Strong electrostatic interactions between titrating sites give rise to complex titration curves. Glu L212 is calculated to have an anomalously broad titration curve, which explains the seemingly contradictory experimental results concerning its pKa. The electrostatic field following electron transfer shifts the average protonation of amino acids near the quinones. The pH dependence of the free energy between Q-AQB and QAQ-B calculated from these shifts is in good agreement with experiment. However, the calculated absolute free energy difference is in severe disagreement (by approximately 230 meV) with the observed experimental value, i.e., electron transfer from Q-A to QB is calculated to be unfavorable. The large stabilization energy of the Q-A state arises from the predominantly positively charged residues in the vicinity of QA in contrast to the predominantly negatively charged residues near QB. The discrepancy between calculated and experimental values for delta G(Q-AQB-->QAQ-B) points to limitations of the continuum electrostatic model. Inclusion of other contributions to the energetics (e.g., protein motion following quinone reduction) that may improve the agreement between theory and experiment are discussed.

Amino Acids↗

PubMed1994

Crystallographic analyses of site-directed mutants of the photosynthetic reaction center from Rhodobacter sphaeroides.

Seven site-directed mutants of the bacterial photosynthetic reaction center (RC) from the 2.4.1 and WS 231 wild-type strains of Rhodobacter sphaeroides have been crystallized and their X-ray diffraction analyzed to resolutions between 3.0 and 4.0 A. The mutations can be divided into four distinct categories: (1) mutations altering cofactor composition that affect electron transfer and quantum yield, His M202-->Leu (M202HL), His L173-->Leu (L173HL), and Leu M214-->His (M214LH); (2) a mutation in the proposed pathway of electron transfer altering electron-transfer kinetics, Tyr M210-->Phe (M210YF); (3) a mutation around the non-heme iron resulting in an iron-less reaction center, His M219-->Cys (M219HC); and (4) mutations around the secondary electron acceptor, a ubiquinone, affecting proton transfer and quinone turnover, Glu L212-->Gln (L212EQ) and Asp L213-->Asn (L213DN). Residues L173 and M202 are within bonding distance of the respective magnesiums of the two bacteriochlorophylls of the BChl special pair, while M214 is close to the bacteriopheophytin on the active A branch of the RC. The L173HL and M202HL crystal structures show that the respective bacteriochlorophylls are replaced with bacteriopheophytins (i.e., loss of magnesium) without significant structural perturbations to the surrounding main-chain or side-chain atoms. In the M214LH mutant, the bacteriopheophytin has been replaced by a bacteriochlorophyll, and the side chain of His M214 is within ligand distance of the magnesium. The M210YF, L212EQ, and L213DN mutants show no significant tertiary structure changes near the mutation sites. The M219HC diffraction data indicate that the overall tertiary structure of the reaction center is maintained in the absence of the non-heme iron.

Chemical Phenomena↗

PubMed1994

Protonation and free energy changes associated with formation of QBH2 in native and Glu-L212-->Gln mutant reaction centers from Rhodobacter sphaeroides.

Formation of the quinol QBH2 in Glu-L212-->Gln mutant [EQ(L212)] reaction centers (RCs) from Rhodobacter sphaeroides was investigated by measuring the proton uptake (using dyes), UV absorption changes, and free energy changes associated with the two-electron reduction of QB. The advantage of using the EQ(L212) RCs for these studies is that the individual protonation steps can be kinetically resolved and analyzed; conclusions reached regarding the mechanism of formation of QBH2 are expected to apply also to native RCs. The proton uptake by EQ(L212) RCs was strongly biphasic: the fast phase was essentially concomitant with the second electron transfer to QB- (approximately 1 ms at pH 7.5); the slow phase was approximately 2000-fold slower. The rate constant of the slow phase depended on the redox state of the primary quinone QA; for QA- the rate constant was larger (i.e., 8-fold at pH 6.0) than for QA. The electron and proton transfers to QB- in EQ(L212) RCs were modeled with a two-step scheme as follows: (1) fast, QA-QB- + H+(1)-->QA(QBH)-; (2) slow, QA(QBH)- + H+(2)-->QAQBH2, where reaction 1 involves concomitant electron transfer and proton uptake [Paddock, M. L., McPherson, P. H., Feher, G., & Okamura, M. Y. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6803-6807]. The stoichiometry of the fast proton uptake associated with the two-electron reduction of QB varied from 1.1 to 1.4 H+/2e- at pH 6.5-8.5, consistent with the uptake of H+(1) plus an additional fractional proton uptake due to amino acid residues whose pKa values are shifted by interactions with the charge of (QBH)-. The total steady-state proton uptake stoichiometry was 2.0 H+/2e- at pH < or = 7.5, consistent with the formation of the quinol QBH2 (reactions 1 and 2). At pH 8.5, the steady-state proton uptake was 1.6 +/- 0.1 H+/2e-, which is consistent with an apparent pKa for H+(2) of approximately 8.5 [McPherson, P. H., Okamura, M. Y., & Feher, G. (1993) Biochim. Biophys. Acta 1144, 309-324]. The proton uptake kinetics indicate that Glu-L212 is a component of the proton transfer chain for H+(2) that connects reduced QB (buried in the RC protein) to the aqueous solvent as proposed previously [Paddock, M. L., Rongey, S. H., Feher, G., & Okamura, M. Y. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6602-6606].(ABSTRACT TRUNCATED AT 400 WORDS)

Glutamine↗

PubMed1994

Pathway of proton transfer in bacterial reaction centers: role of aspartate-L213 in proton transfers associated with reduction of quinoneto dihydroquinone.

The role of Asp-L213 in proton transfer to reduced quinone QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Asp with residues having different proton donor properties. Reaction centers (RCs) with Asn, Leu, Thr, and Ser at L213 had greatly reduced (approximately 6000-fold) proton-coupled electron transfer [kAB(2)] and proton uptake rates associated with the second electron reduction of QB (QA- QB- + 2H(+)-->QAQBH2) compared to native RCs. RCs containing Glu at L213 showed faster (approximately 90-fold) electron and proton transfer rates than the other mutant RCs but were still reduced (approximately 70-fold) compared with native RCs. These results show that kAB(2) is larger when a carboxylic acid occupies the L213 site, consistent with the proposal that Asp-L213 is a component of a proton transfer chain. The reduced kAB(2) observed with Glu versus Asp at L213 suggests that Asp at L213 is important for proton transfer for some other reason in addition to its proton transfer capabilities. Glu-L213 is estimated to have a higher apparent pKa (pKa > or = 7) than Asp-L213 (pKa < or = 4), as indicated by the slower rate of charge recombination (D+QAQB(-)-->DQAQB) in the mutant RCs. The importance of the pKa and charge of the residue at L213 for proton transfer are discussed. Based on these studies, a model for proton transfer is proposed in which Asp-L213 contributes to proton transfer in native RCs in two ways: (1) it is a component of a proton transfer chain connecting the buried QB molecule with the solvent and/or (2) it provides a negative charge that stabilizes a proton on or near QB.

Aspartic Acid↗

PubMed1993

Light-induced proton uptake by photosynthetic reaction centers from Rhodobacter sphaeroides R-26.1. II. Protonation of the state DQAQB2-.

Proton uptake associated with the two-electron reduction of QB was investigated in reaction centers (RCs) from Rhodobacter sphaeroides R-26.1 using pH-sensitive dyes. An uptake of two protons was observed at pH < or = 7.5, consistent with the formation of the dihydroquinone QBH2. At higher pH, the proton uptake decreased with an apparent pKa of approx. 8.5, i.e., to 1.5 H+/2 e- at pH 8.5. A molecular model is presented in which the apparent pKa is due to the protonation of either the carbonyl oxygen on QB or of an amino acid residue near QB (e.g., His-L190). Experimental evidence in favor of the protonation of the oxygen is discussed. The kinetics of the electron transfer from QA-QB- to QAQB2- and the associated proton uptake were compared at several pH values and temperatures. At pH 8.5 (21.5 degrees C) the rate constants for the proton uptake and electron transfer are the same within the precision of the measurement. At lower pH, the proton uptake rate constant is smaller than that for electron transfer. The difference between the rate constants is temperature dependent, i.e., it varies from 12 +/- 4% at 21.5 degrees C (pH 7.5) to 28 +/- 4% at 4.0 degrees C (pH 7.5). We show that the kinetics can be explained by a previously proposed model (Paddock, M. L., McPherson, P. H., Feher, G. and Okamura, M. Y. (1990) Proc. Natl. Acad. Sci. USA 87, 6803-6807) in which the uptake of two protons by doubly reduced QB occurs sequentially, one concomitant with and the other after electron transfer.

Amino Acids↗

PubMed1993

Pathway of proton transfer in bacterial reaction centers: second-site mutation Asn-M44-->Asp restores electron and proton transfer in reaction centers from the photosynthetically deficient Asp-L213-->Asn mutant of Rhodobacter sphaeroides.

Site-directed mutagenesis of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides has shown Asp-213 of the L subunit (Asp-L213) to be important for photosynthetic viability. Replacement of Asp-L213 with Asn resulted in a photosynthetically deficient mutant, due to the 10(4)-fold slower rate for the proton-coupled electron transfer reaction QA-QB- + 2H+-->QAQBH2 (k(2)AB). The detrimental effect of Asn-L213 is surprising since RCs from Rhodopseudomonas viridis, Rhodospirillum rubrum, and Chloroflexus aurantiacus have Asn at the homologous position. However, RCs from these bacteria have an Asp located near QB (the secondary quinone acceptor) at the position homologous to Asn-M44 in Rb. sphaeroides which might function in place of Asp-L213. To test this conjecture a "viridis-like" structure was introduced into Rb. sphaeroides by replacing Asp-L213 with Asn and Asn-M44 with Asp. The RCs from this double mutant displayed near-native rates for the electron transfer reaction k(2)AB and restored photosynthetic competence. The rates for the first electron transfer reaction QA-QB-->QAQB- (k(1)AB) and charge recombination D+QAQB--->DQAQB (kBD) were also restored to near-native values. These results indicate that Asp at either the L213 or the M44 site near QB can provide a pathway for rapid proton transfer and explain why Asp-L213 need not be conserved in different photosynthetic bacteria. To test further the effect of Asp at M44 on electron and proton transfer to QB a mutant containing Asp at both L213 and M44 was constructed. The RCs from this mutant (Asn-M44-->Asp) exhibited faster proton-coupled electron transfer to QB-. The increased rate of proton-coupled electron transfer (k(2)AB) in the presence of negatively charged Asp residues near QB suggests the role of an Asp near QB as (i) a proton donor group in the proton transfer chain and/or (ii) a negatively charged residue stabilizing proton transfer to reduced QB.

Amino Acid Sequence↗

PubMed1992

Proton transfer in reaction centers from photosynthetic bacteria.

Proton transfer in the bacterial RC associated with the reduction of the bound QB to the dihydroquinone is an important step in the energetics of photosynthetic bacteria. The binding of two protons by the quinone is associated with the transfer of the second electron to QB at a rate of ca. 10(3) s-1 (pH 7). Mutation of three protonatable residues, GluL212, SerL223, and AspL213, located near QB to nonprotonatable residues (Gln, Ala, and Asn, respectively) resulted in large reductions (by 2 to 3 orders of magnitude) in the rate or proton transfer to QB. These mutations can be grouped into two classes: those that blocked both proton transfer and electron transfer (SerL223, and AspL213) and those that blocked only proton transfer (GluL212). These results were interpreted in terms of a pathway for proton transport in which uptake of the first proton, required for the transfer of the second electron, occurs through a pathway involving AspL213 and SerL223. Uptake of the second proton, which follows electron transfer, occurs through a pathway involving GluL212 and possibly AspL213. Acidic residues near QB affect electron transfer rates via electrostatic interactions. One residue, with a pKa of ca. 10 interacting strongly with the charge on QB (delta pKa greater than 2), was shown to be GluL212. A second residue with a pKa of ca. 6, which interacts more weakly with the charge on QB (delta pK approximately 1), could be either AspL210 or AspL213. Several possible mechanisms for proton transfer are consistent with the observed experimental results and proposed proton pathways. These involve proton transfers from individual amino acid residues or internal water molecules either as single steps or in a concerted fashion. The determination of the dominant mechanism will require evaluation of the energetics of the various steps.

Bacteria↗

PubMed1991

Protonation of interacting residues in a protein by a Monte Carlo method: application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides.

We used Monte Carlo methods to treat statistical problem of electrostatic interactions among many titrating amino acids and applied these methods to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides, including all titrating sites. We computed the average protonation of residues as a function of pH from an equilibrium distribution of states generated by random sampling. Electrostatic energies were calculated from a finite difference solution to the linearized Poisson-Boltzmann equation using the coordinates from solved protein structures. For most calculations we used the Metropolis algorithm to sample protonation states; for strongly coupled sites, we substantially reduced sampling errors by using a modified algorithm that allows multiple site transitions. The Monte Carlo method agreed with calculations for a small test system, lysozyme, for which the complete partition function was calculated. We also calculated the pH dependence of the free energy change associated with electron transfer from the primary to the secondary quinone in the photosynthetic reaction center. The shape of the resulting curve agreed fairly well with experiment, but the proton uptake from which the free energy was calculated agreed only to within a factor of two with the observed values. We believe that this discrepancy resulted from errors in the individual electrostatic energy calculations rather than from errors in the Monte Carlo sampling.

Electron Transport↗

PubMed1990

Studies of crystal growth mechanisms of proteins by electron microscopy.

We have used electron microscopy to examine the surfaces of lysozyme crystals and deduce mechanisms of crystal growth. We find that growth occurs by a lattice defect mechanism at low supersaturation and by two-dimensional nucleation at high supersaturation. Step velocities and two-dimensional nucleation rates are obtained, and their dependence on supersaturation is compared with theory. Some features of the observed surface structure can be related to the specific topology and strengths of the bonds in the P4(3)2(1)2 lattice. Preliminary results on the early stages of nucleation and the phenomenon of cessation of growth are presented.

Crystallization↗

PubMed1990

Electron paramagnetic resonance investigation of photosynthetic reaction centers from Rhodobacter sphaeroides R-26 in which Fe2+ was replaced by Cu2+. Determination of hyperfine interactions and exchange and dipole-dipole interactions between Cu2+ and QA-.

We report electron paramagnetic resonance (EPR) experiments in frozen solutions of unreduced and reduced photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides R-26 in which Fe2+ has been chemically replaced by the isotope 65Cu2+. Samples in which the primary quinone acceptor QA is unreduced (Cu2+QA:RCs) give a powder EPR spectrum typical for Cu2+ having axial symmetry, corresponding to a d(x2 - y2) ground state orbital, with g values g parallel = 2.314 +/- 0.001 and g perpendicular = 2.060 +/- 0.003. The spectrum shows a hyperfine structure for the nuclear spin of copper (65I = 3/2) with A parallel = (-167 +/- 1) x 10(-4) cm-1 and /A perpendicular/ = (16 +/- 2) x 10(-4) cm-1, and hyperfine couplings with three nitrogen ligands. This has been verified in samples containing the naturally occurring 14N isotope (l = 1), and in samples where the nitrogen ligands to copper were replaced by the isotope 15N (l = 1/2). We introduce a model for the electronic structure at the position of the metal ion which reflects the recently determined three-dimensional structure of the RCs of Rb. sphaeroides (Allen, J. P., G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1987. Proc. Natl. Acad. Sci. USA. 84:5730: Allen, J. P., G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1988. Proc. Natl. Acad. Sci. USA, 85:8487) as well as our EPR results. In this model the copper ion is octahedrally coordinated to three nitrogens from histidine residues and to one carboxylate oxygen from a glutamic acid, forming a distorted square in the plane of the d(x2 = y2) ground state orbital. It is also bound to a nitrogen of another histidine and to the other carboxylate oxygen of the same glutamic acid residue, in a direction approximately normal to this plane. The EPR spectrum changes drastically when the quinone acceptor QA is chemically reduced (Cu2+QA-:RCs); the change is due to the exchange and dipole-dipole interactions between the Cu2+ and QA- spins. A model spin Hamiltonian proposed for this exchange coupled cooper-quinone spin dimer accounts well for the observed spectra. From a comparison of the EPR spectra of the Cu2+QA:RC and CU2+QA-:RC complexes we obtain the values /J0/ = (0.30 +/- 0.02) K for the isotropic exchange coupling, and /d/ = (0.010 +/- 0.002) K for the projection of the dipole-dipole interaction tensor on the symmetry axis of the copper spin. From the EPR experiments only the relative signs of J0 and d can be deduced; it was determined that they have the same sign. The magnitude of the exchange coupling calculated for Cu2+QA-:RC is similar to that observed for the Fe2+QA-:RC complex (J0 = -0.43K). The exchange coupling is discussed in terms of the superexchange paths connecting the Cu2+ ion and the quinone radical using the structural data for the RCs of Rb. sphaeroides. From the value of the dipole-dipole interaction, d, we determined R approximately 8.4 A for the weighted distance between the metal ion and the quinone in reduced RCs, which is to be compared with 10 A obtained from x-ray analysis of unreduced RCs. This points to a shortening of the Cu2+ -QA- distance upon reduction of the quinone, as has been proposed by Allen et al. (1988).

Amino Acid Sequence↗