Raman signatures of ligand binding and allosteric conformation change in hexameric insulin.

Hexameric insulin is an allosteric protein that undergoes transitions between three conformational states (T(6), T(3)R(3), and R(6)). These allosteric states are stabilized by the binding of ligands to the phenolic pockets and by the coordination of anions to the His B10 metal sites. Raman difference (RD) spectroscopy is utilized to examine the binding of phenolic ligands and the binding of thiocyanate, p-aminobenzoic acid (PABA), or 4-hydroxy-3-nitrobenzoic acid (4H3N) to the allosteric sites of T(3)R(3) and R(6). The RD spectroscopic studies show changes in the amide I and III bands for the transition of residues B1-B8 from a meandering coil to an alpha helix in the T-R transitions and identify the Raman signatures of the structural differences among the T(6), T(3)R(3), and R(6) states. Evidence of the altered environment caused by the approximately 30 A displacement of phenylalanine (Phe) B1 is clearly seen from changes in the Raman bands of the Phe ring. Raman signatures arising from the coordination of PABA or 4H3N to the histidine (His) B10 Zn(II) sites show these carboxylates give distorted, asymmetric coordination to Zn(II). The RD spectra also reveal the importance of the position and the type of substituents for designing aromatic carboxylates with high affinity for the His B10 metal site.

Characterization of carotenoid and chlorophyll photooxidation in photosystem II.

Photosystem II (PSII) contains two accessory chlorophylls (Chl(Z), ligated to D1-His118, and Chl(D), ligated to D2-His117), carotenoid (Car), and heme (cytochrome b(559)) cofactors that function as alternate electron donors under conditions in which the primary electron-donation pathway from the O(2)-evolving complex to P680(+) is inhibited. The photooxidation of the redox-active accessory chlorophylls and Car has been characterized by near-infrared (near-IR) absorbance, shifted-excitation Raman difference spectroscopy (SERDS), and electron paramagnetic resonance (EPR) spectroscopy over a range of cryogenic temperatures from 6 to 120 K in both Synechocystis PSII core complexes and spinach PSII membranes. The following key observations were made: (1) only one Chl(+) near-IR band is observed at 814 nm in Synechocystis PSII core complexes, which is assigned to Chl(Z)(+) based on previous spectroscopic studies of the D1-H118Q and D2-H117Q mutants [Stewart, D. H., Cua, A., Chisholm, D. A., Diner, B. A., Bocian, D. F., and Brudvig, G. W. (1998) Biochemistry 37, 10040-10046]; (2) two Chl(+) near-IR bands are observed at 817 and 850 nm in spinach PSII membranes which are formed with variable relative yields depending on the illumination temperature and are assigned to Chl(Z)(+), and Chl(D)(+), respectively; (3) the Chl and Car cation radicals have significantly different stabilities at reduced temperatures with Car(+) decaying much faster; (4) in Synechocystis PSII core complexes, Car(+) decays by recombination with Q(A)(-) and not by Chl(Z)/Chl(D) oxidation, with multiphasic kinetics that are attributed to an ensemble of protein conformers that are trapped as the protein is frozen; and (5) in spinach PSII membranes, Car(+) decays mainly by recombination with Q(A)(-), but also partly by formation of the 850 nm Chl cation radical. The greater stability of Chl(Z)(+) at low temperatures enabled us to confirm that resonance Raman bands previously assigned to Chl(Z)(+) are correctly assigned. In addition, the formation and decay of these cations provide insight into the alternate electron-donation pathways to P680(+).

Relationship between altered structure and photochemistry in mutant reaction centers in which bacteriochlorophyll replaces the photoactive bacteriopheophytin.

Qy-excitation resonance Raman (RR) spectra are reported for two mutant reaction centers (RCs) from Rhodobacter capsulatus in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated beta. The pigment change in both mutants is induced via introduction of a histidine residue near the photoactive cofactor. In one mutant, L(M212)H, the histidine is positioned over the core of the cofactor and serves as an axial ligand to the Mg+2 ion. In the other mutant, F(L121)H/F(L97)V, the histidine is positioned over ring V of the cofactor, which is nominally too distant to permit bonding to the Mg+2 ion. The salient observations are as follows: (1) The beta cofactor in F(L121)H/F(L97)V RCs is a five-coordinate BChl molecule. However, there is no evidence for the formation of a Mg-His bond. This bond is either much weaker than in the L(M212)H RCs or completely absent, the latter implying coordination by an alternative ligand. The different axial ligation for beta in the F(L121)H/F(L97)V versus L(M212)H RCs in turn leads to different conformations of the BChl macrocycles. (2) The C9-keto group of beta in F(L121)H/F(L97)V RCs is free of hydrogen bonding interactions, unlike the L(M212)H RCs in which the C9-keto of beta is hydrogen bonded to Glu L104. The interactions between other peripheral substituents of beta and the protein are also different in the F(L121)H/F(L97)V RCs versus L(M212)H RCs. Accordingly, the position and orientation of beta in the protein is different in the two beta-containing RCs. Nonetheless, previous studies have shown that the primary electron transfer reactions are very similar in the two mutants but differ in significant respects compared to wild-type RCs. Collectively, these observations indicate that changes in the conformation of a photoactive tetrapyrrole macrocycle or its interactions with the protein do not necessarily lead to significantly perturbed photochemistry and do not underlie the altered primary events in beta-type RCs.

Functionalized de novo designed proteins: mechanism of proton coupling to oxidation/reduction in heme protein maquettes.

Proton exchange with aqueous media coupled to heme oxidation/reduction is commonly seen but not understood in natural cytochromes. Our synthetic tetrahelix bundle heme protein maquettes successfully reproduce natural proton coupling to heme oxidation/reduction. Potentiometry reveals major pK shifts from 4.2 to 7.0 and from 9.4 to 10.3 in the maquette-associated acid/base group(s) upon heme reduction. Consequently, a 210 mV decrease in the heme redox potential is observed between the two extremes of pH. Potentiometry with resonance Raman and FTIR spectroscopy performed over a wide pH range strongly implicates glutamate side chains as the source of proton coupling below pH 8.0, whereas lysine side chains are suggested above pH 8.0. Remarkably, the pK values of several glutamates in the maquette are elevated from their solution value (4.4) to values as high as 7.0. It is suggested that these glutamates are recruited into the interior of the bundle as part of a structural rearrangement that occurs upon heme binding. Glutamate to glutamine variants of the prototype protein demonstrate that removal of the glutamate closest to the heme diminishes but does not abolish proton exchange. It is necessary to remove additional glutamates before pH-independent heme oxidation/reduction profiles are achieved. The mechanism of redox-linked proton coupling appears to be rooted in distributed partial charge compensation, the magnitude of which is governed by the dielectric distance between the ferric heme and acid/base side chains. A similar mechanism is likely to exist in native redox proteins which undergo charge change upon cofactor oxidation/reduction.

Identification of histidine 118 in the D1 polypeptide of photosystem II as the axial ligand to chlorophyll Z.

Chlorophyll Z (ChlZ) is a redox-active chlorophyll (Chl) which is photooxidized by low-temperature (<100 K) illumination of photosystem II (PSII) to form a cation radical, ChlZ+. This cofactor has been proposed to be an "accessory" Chl in the PSII reaction center and is expected to be buried in the transmembrane region of the PSII complex, but the location of ChlZ is unknown. A series of single-replacement site-directed mutants of PSII were made in which each of two potentially Chl-ligating histidines, D1-H118 or D2-H117, was substituted with amino acids which varied in their ability to coordinate Chl. Assays of the wild-type and mutant strains showed parallel phenotypes for the D1-118 and D2-117 mutants: noncoordinating or poorly coordinating residues at either position decreased photosynthetic competence and impaired assembly of PSII complexes. Only the mutants substituted with glutamine (D1-H118Q and D2-H117Q) had phenotypes comparable to the wild-type strain. The ChlZ+ cation was characterized by low-temperature electron paramagnetic resonance (EPR), near-infrared (IR) absorbance, and resonance Raman (RR) spectroscopies in wild-type, H118Q, and H117Q PSII core complexes. The quantum yield of ChlZ+ formation is the same (approximately 2.5% per saturating flash at 77 K) for wild-type, H118Q, and H117Q, indicating that its efficiency of photooxidation is unchanged by the mutations. Similarly, the EPR and near-IR absorbance spectra of ChlZ+ are insensitive to the mutations made at D1-118 and D2-117. In contrast, the RR signature of ChlZ+ in H118Q PSII, obtained by selective near-IR excitation into the ChlZ+ cation absorbance band, is significantly altered relative to wild-type PSII while the RR spectrum of ChlZ+ in the H117Q mutant remains identical to wild-type. Shifts in the RR spectrum of ChlZ+ in H118Q reflect a change in the structure of the Chl ring, most likely due to a perturbation of the core size and/or extent of doming caused by a change in the axial ligand to Mg(II). Thus, we conclude that the axial ligand to ChlZ is H118 of the D1 polypeptide. Furthermore, we propose that H117 of the D2 polypeptide is the ligand to a homologous redox-inactive accessory Chl which we term ChlD. The Chl Z and D terminology reflects the 2-fold structural symmetry of PSII which is apparent in the redox-active tyrosines, YZ and YD, and the active/inactive branch homology of the D1/D2 polypeptides with the L/M polypeptides of the bacterial reaction center.

Resonance raman characterization of reaction centers with an Asp residue near the photoactive bacteriopheophytin.

Qy-excitation resonance Raman (RR) studies are reported for a series of Rhodobacter capsulatus reaction centers (RCs) containing mutations at L-polypeptide residue 121 near the photoactive bacteriopheophytin (BPhL). The studies focus on the electronic/structural perturbations of BPhL induced by replacing the native Phe with an Asp residue. Earlier work has shown that the electron-transfer properties of F(L121)D RCs are closely related to those of RCs in which BPhL is replaced by bacteriochlorophyll (BChl) (beta-type RCs) or by pheophytin. In addition to the F(L121)D single mutant, RR studies were performed on the F(L121)D/E(L104)L double mutant, which additionally removes the hydrogen bond between BPhL and the native Glu L104 residue. The vibrational signatures of BPhL in the single and double mutants containing Asp L121 are compared with one another and with those of BPhL in both wild-type and F(L121)L RCs. The replacement of the aromatic Phe residue with Leu has no discernible effect on the vibrational properties of BPhL, a finding in concert with the previously reported absence of an effect of the mutation on the electron-transfer characteristics of the RC. In contrast, replacement of Phe with Asp significantly perturbs the vibrational characteristics of BPhL, and in a manner most consistent with Asp L121 being deprotonated and negatively charged. The negative charge of the carboxyl group of Asp L121 interacts with the pi-electron system of BPhL in a relatively nonspecific fashion, diminishing the contribution of charge-separated resonance forms of the C9-keto group to the electronic structure of the cofactor. The presence of a negative charge near BPhL is consistent with the known photochemistry of F(L121)D RCs, which indicates that the free energy of P+BPhL- is substantially higher than in wild-type RCs.

Disruption of the heme iron-proximal histidine bond requires unfolding of deoxymyoglobin.

The unfolding behavior of 10 different distal heme pocket mutants of sperm whale deoxymyoglobin (deoxyMb) has been investigated. The effects of distal histidine (His 64) replacement were the primary focus; however, mutations at Leu 29, Val 68, and Ile 107 were also examined. Formation of the spectroscopically distinguishable heme intermediate (I') of deoxyMb was tracked as a function of pH and guanidinium chloride (GdmCl) concentration. The appearance of this intermediate signals cleavage of the iron-proximal histidine (His 93) bond. The key observations are as follows. (1) None of the distal heme pocket mutations investigated alter the nature of the heme intermediates that are formed under low pH unfolding conditions. (2) Unfolding of deoxyMb at high concentrations of GdmCl proceeds through the same heme intermediates that occur under low pH conditions. (3) The rate of the iron-histidine bond cleavage in an acidic medium is dramatically slowed when large hydrophobic residues (Leu and Phe) replace the distal histidine, whereas there is little correlation between the polarity of the residue at position 64 and the rate of denaturation by GdmCl. (4) However, apolar residues at position 64 enhance significantly the equilibrium resistance of deoxyMb to iron-histidine bond cleavage under both low pH and high GdmCl unfolding conditions. There is a direct correlation between the equilibrium pH and GdmCl values for maximum intermediate formation and the stabilities of the corresponding apoproteins. Collectively, these observations suggest that substantial unfolding of deoxyMb is required for Fe(II)-His 93 bond cleavage. Unlike the situation for aquometMb, heme loss from deoxyMb is not driven by protonation of the proximal histidine ligand. Instead, the process is mediated by more global unfolding of the protein that leads to solvation of the prosthetic group.

Resonance Raman characterization of reaction centers in which bacteriochlorophyll replaces the photoactive bacteriopheophytin.

Qy-excitation resonance Raman (RR) spectra are reported for two mutant reactions centers (RCs) from Rhodobacter sphaeroides in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated by beta L. One mutation, (M)L214H, yields the pigment change via introduction of a histidine residue at position M214. The other mutation, (M)L214H/(L)-E104V, removes the putative hydrogen bond between beta L and the native glutamic acid residue at position L104. The vibrational signatures of the beta L cofactors of the mutants are compared with one another and with those of the accessory BChls (BChlL,M) in both beta-mutant and wild-type RCs. The spectroscopic data reveal the following: (1) The beta L cofactor is a five-coordinate BChl molecule with a histidine axial ligand. The conformation of beta L and the strength of the Mg-histidine bond are very similar to that of BChlL,M. (2) The beta L cofactor is oriented in the protein pocket in a manner similar to that of BPhL of wild-type. (3) The beta L cofactor of the (M)L214H mutant forms a hydrogen bond with glutamic acid L104 via the C9-keto group of the macrocycle. The strength of this hydrogen bond is identical to that formed between this protein residue and the C9-keto group of BPhL in wild-type. (4) The hydrogen bonding interaction at the C9-keto site induces secondary cofactor-protein interactions which involve the C2a-acetyl and Cb-alkyl substituent groups. Collectively, the vibrational signatures of beta L indicate that its intrinsic physicochemical properties are very similar to those of BChlL. Consequently, the initial charge-separated intermediate in beta-type RCs is best characterized as a thermal/quantum mechanical admixture of P+ beta L- and P+ BChlL-(P is the primary electron donor), as originally proposed by Kirmaier et al. [(1995) J. Phys. Chem. 99, 8903-8909].

Qy-excitation resonance Raman spectra of chlorophyll a and bacteriochlorophyll c/d aggregates. Effects of peripheral substituents on the low-frequency vibrational characteristics.

Low-frequency (80-700 cm-1) Qy-excitation resonance Raman (RR) spectra are reported for thin-solid-film aggregates of several chlorophyll (Chl) a and bacteriochlorophyll (BChl) c/d pigments. The pigments include Chl a, pyrochlorophyll a (PChl a), methylpyrochloropyllide a (MPChl a), methylbacteriochloropyllide d (MBChl d), [E,M] BChl cS, [E,E] BChl cF, and [P,E] BChl cF. The BChl c/d's are the principal constituents of the chlorosomal light-harvesting apparatus of green photosynthetic bacteria. Together, the various Chl a's and BChl c/d's represent a series in which the peripheral substituent groups on the chlorin macrocycle are varied in systematic fashion. All of the Chl a and BChl c/d aggregates exhibit rich low-frequency vibrational patterns. In the case of the BChl c/d's, certain modes in the very low-frequency region (100-200 cm-1) experience exceptionally strong Raman intensity enhancements. The frequencies of these modes are qualitatively similar to those of oscillations observed in femtosecond optical experiments on chlorosomes. The RR data indicate that the low-frequency vibrations are best characterized as intramolecular out-of-plane deformations of the chlorin macrocycle rather than intermolecular modes. The coupling of the out-of-plane modes in turn implies that the Qy electronic transition(s) of the aggregate have out-of-plane character. The RR spectra of the BChl c/d's also reveal that the nature of the alkyl substituents at the 8 and 12 positions of the macrocycle plays an important role in determining the detailed features of the low-frequency vibrational patterns. The frequencies of the modes are particularly sensitive to larger substituent groups whose conformations may be more easily perturbed in the tightly packed aggregates. These factors also make aggregates of pigments containing larger substituents more susceptible to structural, electronic, and vibrational inhomgeneities. Collectively, the RR studies of the various pigments delineate the factors which influence the low-frequency vibrational characteristics of chlorosomal aggregates.

Structural and electronic properties of the heme cofactors in a multi-heme synthetic cytochrome.

Resonance Raman, absorption, and electron paramagnetic resonance spectra are reported for a water soluble, synthetic cytochrome. The protein is a variant of the cytochrome beta maquette described by Robertson et al. [Robertson, D. E., et al. (1995) Nature 368, 425-432] and is composed of 62 amino acid residues arranged in a di-alpha-helical unit which dimerizes in solution to form a four-helix bundle. Each di-alpha-helical unit contains histidine residues at the 10,10' positions which serve as ligands to the hemes. When protoheme IX is incorporated, both hemes in the dimer are bis-ligated and low spin. The two hemes are inequivalent with respect to both binding affinity and redox properties. To investigate the properties of the heme cofactors, spectroscopic studies were conducted on peptides reconstituted with protoheme IX (PHa) and several related variants. These hemes include 2-vinyldeuteroheme (2-VDH), 4-vinyldeuteroheme (4-VDH), protoheme III (PHs), and 1-methyl-2-oxomesoheme XIII (2-OMH). Collectively, the spectroscopic studies reveal the following: (1) 2-VDH, 4-VDH, and 2-OMH bind to the protein and form bis-ligated low-spin complexes similar to PHa. The structures of the two hemes in the dimers are identical as are the immediate protein environments around the bound cofactors. These results indicate that the redox inequivalence of the two hemes is due to heme-heme electronic interactions rather than structural and/or environmental differences between the two cofactors. (2) The two hemes in the dimer are arranged in a edge-to-edge arrangement wherein the oxo group (2-OMH) or the vinyl group(s) are in the hydrophobic interface between the two units which comprise the dimer. The propionic acid tails point outward toward the hydrophilic region and extend into the solvent. (3) The PHs protein differs from the other synthetic proteins in that it contains one pentacoordinate, high-spin and one hexacoordinate, low-spin heme rather than two hexacoordinate low-spin cofactors. The open coordination site on the high-spin heme is inaccessible to exogenous imidazole but readily bind cyanide, suggesting that the alpha-helix containing the unbound histidine is nearby and partially shields the coordination site. The high-spin heme converts to low-spin at low-temperature, presumably via binding of the histidine residue on this nearby alpha-helix. It is suggested that the different behavior observed for the PHs protein is due to the fact that this heme is symmetric with respect to rotation about the alpha,gamma-axis of the macrocycle which bisects the meso-carbons between the vinyl groups and propionic acid residues. This symmetry precludes rotational isomerism about the alpha,gamma-axis to establish an unhindered fit. In contrast, all the other hemes examined contain at least one substituent smaller than a vinyl group which together with the fact that two different alpha,gamma-rotational isomers are possible for each heme in the dimer could allow these hemes to avoid the like-substituent--like-substituent heme--heme interactions of PHs. The propensity to avoid such interactions could explain the inequivalent binding properties of the two hemes in the dimer. For the PHs protein wherein these these interactions cannot be mitigated by rotation of the heme, other rearrangements of the protein must occur. These rearrangements could force the second-bound heme to assume a high-spin configuration.

Resonance Raman characterization of H(M200)L mutant reaction centers from Rhodobacter capsulatus. Effects of heterodimer formation on the structural and electronic properties of the cofactors.

Resonance Raman (RR) spectra are reported for photosynthetic reactions centers (RCs) from the H(M200)L mutant of Rhodobacter capsulatus. In this mutant, the histidine residue which ligates the M-side bacteriochlorophyll (BCh) of the special pair primary donor (P) of wild-type RCs is replaced by a noncoordinating leucine. This results in the formation of a heterodimer primary donor (D) in which a bacteriopheophytin (BPh) replaces the M-side BCh. The RR data for the H(M200)L mutant were acquired at a large number of excitation wavelengths which span the B, Qx, and Qy absorption bands of the various bacteriochlorin cofactors in the RC. For comparison, spectra were also acquired for wild-type RCs at the same excitation wavelengths. The RR data obtained for the mutant indicate that heterodimer formation induces a variety of changes in the structural and electronic properties of the cofactors in the RC. These perturbations extend beyond the primary donor and include one of the two accessory BChs. Collectively, the RR studies indicate the following: (1) The structure of the single BCh cofactor in D [DL(BCh)] is different from that of either of the two BChs in P. However, DL(BCh) is more similar to PL than to PM. The PM cofactor is conformationally more distorted than either PL or DL(BCh). (2) The structure of the BPh cofactor in D [DM(BPh)] is similar to that of the other two BPhs in the RC. However, the frequency of the C9-keto carbonyl mode of DM(BPh) is anomalously low (1678 cm-1), as is also the case for PM. The vibrational characteristics of the C9-keto carbonyl vibrations of DM(BPh)/PM versus DL(BCh)/PL are consistent the notion that dielectric effects govern the frequency of the mode and that the effective dielectric constant is different on the L- versus M-sides of the primary donor. (3) Heterodimer formation perturbs the structural and electronic properties of one of the two accessory BChs (most likely BChL) in the RC. These perturbations are manifested as upshifts in the ring skeletal-mode frequencies and a blue-shift in the Qx absorption band (from 600 to 580 nm). The fact that heterodimer formation perturbs one of the accessory BChs suggests that global structural rearrangements occur in the protein matrix when the ligand to a cofactor in the primary donor is removed. (4) For both the H(M200)L mutant and wild-type RCs, oxidation of the primary donor significantly affects the RR cross section of the carotenoid.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of the iron-carbonyl stretch in distal histidine mutants of carbonmonoxymyoglobin.

Soret-excitation resonance Raman (RR) spectra are reported for six distal histidine mutants of carbonmonoxymyoglobin including H64A, H64V, H64L, H64I, H64W, and H64W/L29F. Based on 13CO isotope shifts, the iron-carbonyl stretching vibrations are unambiguously identified. The correct assignment of these modes eliminates the differences in the conformational substate occupations predicted by the RR versus IR data.

Acid-induced transformations of myoglobin. Characterization of a new equilibrium heme-pocket intermediate.

The pH dependence of the absorption and resonance Raman (RR) spectra of the deoxy and met forms of myoglobin (Mb) has been examined in detail. The spectral data were acquired at a number of different pHs (12) in the 2.6-7.6 range. RR spectra were obtained for both the low- and high-frequency regions by using a variety of excitation wavelengths ranging from the UV to the green. The data obtained for deoxyMb indicate that a spectroscopically distinct intermediate (I') exists at equilibrium in the pH 3.5-4.5 range. The I'-form of metMb could not be identified. The Soret absorption maximum of the I'-form of deoxyMb is at approximately 426 nm compared with the value of 435 observed for the native (N) form and 383 nm observed for the so-called unfolded (U') form which occurs in the pH 2.6-3.5 range. The absorption and vibrational spectra of the I'-form of deoxyMb observed at equilibrium are very similar to those of the intermediate that appears within a few milliseconds in pH-jump experiments. The RR data indicate that the structure of the heme group in the I'-form is distinctly different from that of either N- or U'-forms. The iron-histidine bond, characteristic of the N-form, is ruptured in both the I'- and U'-forms as is evidenced by the absence of the RR band due to the stretching vibration of this unit. In the I'-form, the histidine ligand is replaced by a relatively strongly bound, exchangeable water molecule. This ligand is absent in the U'-form. The aquo ligand of the five-coordinate heme in the I'-form is identified by a RR band at 411 cm-1 which undergoes a 15-17 cm-1 downshift in deuteriated buffer solutions. In contrast, none of the RR bands of the N- and U'-forms exhibit any significant isotope sensitivity. The properties of the I'-form and the conditions under which it is generated strongly suggest that this form corresponds to the molten globule intermediate of apoMb.

Qy-excitation resonance Raman scattering from the special pair in Rhodobacter sphaeroides reaction centers. Implications for primary charge separation.

Qy-excitation resonance Raman (RR) spectra are reported for reaction centers (RCs) from Rhodobacter sphaeroides 2.4.1. The RR spectra were acquired for both chemically reduced and oxidized RCs at 25 and 201 K by using a variety of excitation wavelengths in the range 800-920 nm. This range spans the Qy absorption bands of the special pair (P) and the accessory bacteriochlorophylls (BChls). The RR studies indicate that both P and the accessory BChls exhibit rich RR spectra in the 30-1800-cm-1 region. For both types of pigments, at least 20 bands are observed in the 30-750-cm-1 range. Although the frequencies of the modes of P and the accessory BChls are different, it is possible to make one-to-one correlations of the bands observed for the two types of pigments. This result suggests that the vibronically active low-frequency modes of P are derived from monomer-like vibrations (although they may be coupled monomer-like modes) rather than being vibrations resulting from the additional degrees of freedom present in the dimer. A plausible set of vibrational assignments for the low-frequency modes of both P and the accessory BChls is proposed on the basis of a semiempirical normal coordinate calculation. Comparison of the RR intensities of the low-frequency modes of P with those of the analogous modes of the accessory BChls indicates that the intensities of the modes of the former pigments are considerably larger than those of the latter. Collectively, the spectral data indicate that a large number of low-frequency modes of P are strongly coupled to the Qy electronic transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pigment-protein interactions on the conformation of the primary electron acceptor in Rhodobacter capsulatus reaction centers.

Resonance Raman spectra are reported for RCs from Rb. capsulatus in which the L104 glutamic acid is replaced by glutamine. The skeletal modes of the primary electron acceptor, BPhL, in these RCs undergo temperature-dependent frequency shifts that are identical to those observed for BPhL in RCs from wild-type. This observation suggests that the strength of the hydrogen bond between the L104 residue and the C9 keto group of BPhL is not a determinant of the temperature-dependent conformation of this pigment.

Resonance Raman studies of genetically modified reaction centers from Rhodobacter capsulatus.

Resonance Raman (RR) spectra are reported for the photosynthetic reaction center (RC) proteins from Rhodobacter capsulatus wild type and the genetically modified systems GluL104----Leu and HisM200----Leu. The spectra were obtained with a variety of excitation wavelengths, spanning the UV, violet, and yellow-green regions of the absorption spectrum, and at temperatures of 30 and 200 K. The RR data indicate that the structures of the bacteriochlorin pigments in RCs from Rb. capsulatus wild type are similar to those in RCs from Rhodobacter sphaeroides wild type. The data also show that the amino acid modifications near the primary electron acceptor (GluL104----Leu) and special pair (HisM200----Leu) perturb only those bacteriochlorin pigments near the site of the mutation and do not influence the structures of the other pigments in the RC. In the case of the GluL104----Leu mutant, elimination of the hydrogen bond to the C9 keto group of BPhL results in frequency shifts of RR bands of certain skeletal modes of the macrocycle. This allows the assignment of bands to the individual BPhL and BPhM pigments. In the case of the HisM200----Leu mutant, in which the special pair is comprised of a bacteriochlorophyll (BChl)-bacteriopheophytin (BPh) heterodimer rather than the BChl2 unit bound in the wild type, certain skeletal vibrations due to the additional BPh unit are identified. The frequencies of these modes are similar to those of the analogous vibrations BPhL and BPhM, which indicates that the structure of the BPh in the heterodimer is not unusual in any discernible way.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature-dependent conformational changes in the bacteriopheophytins of Rhodobacter sphaeroides reaction centers.

Resonance Raman (RR) spectra are reported for the photosynthetic reaction center (RC) protein from Rhodobacter sphaeroides 2.4.1. The spectra were obtained with a variety of excitation wavelengths, spanning the UV, violet, and yellow-green regions of the absorption spectrum, and at a number of temperatures ranging from 30 to 270 K. The RR data indicate that the frequencies of certain vibrational modes of the bacteriochlorin pigments in the RC shift with temperature. These shifts are reversible and do not depend on external factors such as solvent or detergent. The acetyl carbonyl bands exhibit the largest shifts with temperature. These shifts are attributed to thermal effects involving the torsional vibrations of the acetyl groups of several (or all) of the bacteriochlorins rather than to specific pigment-protein interactions. The frequency of the structure-sensitive skeletal mode near 1610 cm-1 of one of the two bacteriopheophytins (BPhs) in the RC is also sensitive to temperature. In contrast, no temperature sensitivity is observed for the analogous modes of the bacteriochlorophylls or other BPhs. Over the range 160-100 K, the skeletal mode of the BPh upshifts by approximately 4 cm-1. This upshift is attributed to a flattening of the macrocycle at low temperatures. It is suggested that the BPh active in the electron-transfer process is the pigment whose structure is temperature dependent. It is further suggested that such structural changes could be responsible in part for the temperature dependence of the electron-transfer rates in photosynthetic RCs.