Structure of the primary electron donor in photosystem I: a resonance Raman study

Low-temperature resonance Raman (RR) spectra have been obtained at resonance with the Soret transition of chlorophyll a in photosystem I particles containing large amounts either of the triplet state of P700 or of its radical cation state. Subtracting these spectra from those of resting reaction centers yielded RR spectra of P700 in its neutral, ground state. These spectra arise from two distinct chlorophyll a molecules differing by the strengths of the bonding interactions assumed by their keto carbonyl groups, the stretching frequencies of which are found at 1655 and 1675 cm-1. The present results rule out previous hypotheses that P700 might have consisted of a single, chemically modified chlorophyll a molecule. Neither of the bonding interactions assumed by the keto carbonyls of the P700 chlorophylls most probably involves chlorophyll-chlorophyll bridging through water molecules, as surmised in the so-called special pair models, but likely consists of H bonds with distinct protein sites. The magnesium atoms of the two P700 chlorophylls are 5-coordinated. Hence, the structural model of P700 provided by the present data is qualitatively the same, in terms of bonding interactions, as that currently accepted for the bacterial primary donor.     

Interaction of the photosystem 1 reaction center with antenna chlorophyll in a pigment-protein complex isolated from pea chloroplasts

Using a specially developed phosporoscopic attachment to spectropolarimeter, light induced spectra of circular dichroism (CD) in region 600-750 nm were measured for a pigment protein complex of photosystem 1 (PC-1) isolated from pea chloroplast (chlorophyll : P700 = 40). Minor components at 672 and 678 nm are observed in light induced spectra besides the components of dimer splitting of P700 Qy transition at 691 and 698 nm. Haussian deconvolution of light induced CD spectra of P700 and low temperature CD spectrum of PC-1 indicates that minor components are due to forms of antenna chlorophylls Chl672 and Chl678, rotational strength of that is changed by 2-4% as a result of P700 oxidation. Long term incubation of PC-1 with Triton X-100 inhibits P700 and destroys longwave optically active chlorophyll forms. A strong relation between dichroic density of 693 nm band in CD spectrum of PC-1 and the value of light induced absorption change at 698 nm could be used to determine P700 concentration on the basis of CD spectrum of PC-1. Such a relation shows that Chl693 is an important component of photo-system 1 reaction center. It is suggested that P700 is not an isolated dimer but it is included in the local complex from 8-10 chlorophyll molecules (Chl672, Chl678, Chl686, Chl693).     

Aggregation of chlorophyll a probed by resonance light scattering spectroscopy.

We report the resonance light scattering (RLS) spectra of chlorophyll a aggregated in a 9:1 solution of formamide and pH 6.8 phosphate buffer. The aggregate formed after 2 h of mixing, referred to as Chl469, shows a strong scattering feature at 469 nm (Soret band) and a much weaker feature at 699 nm (Qy band). A kinetic investigation confirmed that the aggregation process is cooperative, and also detected one intermediate (Chl458) with a strong RLS spectrum but only a weak CD spectrum. We propose that aggregation proceeds via at least three steps: 1) formation of a nucleating species, probably a dimer of chlorophylls; 2) formation of large aggregates with little or no secondary structure (e.g., Chl458); and 3) conformational change to form helical aggregate (Chl469).     

Resonance Raman spectra of chlorophylls dissolved in liquid crystal matrices. I. The interaction between chlorophylls and a liquid crystalline MBBA + EBBA mixture

The resonance Raman (RR) spectra of chlorophyll (Chl) a, Chl b and pheophytin a dissolved in a liquid-crystalline MBBA + EBBA mixture were measured. The RR frequencies of chlorophylls in the liquid crystal (LC) are compared with those of solutions of various chlorophyll monomers and aggregates taken from the literature. It is concluded that Chl a and Chl b in LC exist largely as the solvated monomers, even at high concentration. The magnesium atoms in both Chl a and Chl b are pentacoordinated.     

Primary donor photo-oxidation in photosystem I: a re-evaluation of (P700(+) - P700) Fourier transform infrared difference spectra.

Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the photo-oxidation of the primary electron donor (P700) in PS I particles from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803. To aid in the interpretation of the spectra, PS I particles from a site-directed mutant of C. reinhardtii, in which the axial histidine ligand (HisA676) was changed to serine, were also studied. A high-frequency (3300-2600 cm(-1)) electronic transition is observed for all PS I particles, demonstrating that P700 is dimeric. The electronic band is, however, species-dependent, indicating some differences in the electronic structure of P700 and/or P700(+) in C. reinhardtii and Synechocystis sp. 6803. For PS I particles from C. reinhardtii, substitution of HisA676 with serine has little effect on the ester carbonyl modes of the chlorophylls of P700. However, the keto carbonyl modes are considerably altered. Comparison of (P700(+) - P700) FTIR difference spectra obtained using PS I particles from the wild type (WT) and the HS(A676) mutant of C. reinhardtii indicates that the mutation primarily exerts its influence on the P700 ground state. The 13(1) keto carbonyls of the chlorophylls of P700 of the wild type absorb at similar frequencies, which has previously made these transitions difficult to resolve. However, for the HS(A676) mutant, the 13(1) keto carbonyl of chlorophyll a or chlorophyll a' of P700 on PsaB or PsaA absorbs at 1703.4 or 1694.2 cm(-1), respectively, allowing their unambiguous resolution. Upon P700(+) formation, in both PS I particles from C. reinhardtii, the higher-frequency carbonyl band upshifts by approximately 14 cm(-1) while the lower frequency carbonyl downshifts by approximately 10 cm(-1). The similarity in the spectra for WT PS I particles from C. reinhardtii and Synechocystis sp. 6803 indicates that a similar interpretation is probably valid for PS I particles from both species. The mutant results allow for an interpretation of the behavior of the 13(1) keto carbonyls of P700 that is different from previous work [Breton, J., Nabedryk, E., and Leibl, W. (1999) Biochemistry 38, 11585-11592], in which it was suggested that 13(1) keto carbonyls of P700 absorb at 1697 and 1639 cm(-1), and upshift by 21 cm(-1) upon cation formation. The interpretation of the spectra reported here is more in line with recent results from ENDOR spectroscopy and high-resolution crystallography.     

Fluorescence and absorption spectroscopy of the weakly fluorescent chlorophyll a in cytochrome b6f of Synechocystis PCC6803.

A spectroscopic characterization of the chlorophyll a (Chl) molecule in the monomeric cytochrome b6f complex (Cytb6f) isolated from the cyanobacterium Synechocystis PCC6803 is presented. The fluorescence lifetime and quantum yield have been determined, and it is shown that Chl in Cytb6f has an excited-state lifetime that is 20 times smaller than that of Chl in methanol. This shortening of the Chl excited state lifetime is not caused by an increased rate of intersystem crossing. Most probably it is due to quenching by a nearby amino acid. It is suggested that this quenching is a mechanism for preventing the formation of Chl triplets, which can lead to the formation of harmful singlet oxygen. Using site-selected fluorescence spectroscopy, detailed information on vibrational frequencies in both the ground and Qy excited states has been obtained. The vibrational frequencies indicate that the Chl molecule has one axial ligand bound to its central magnesium and accepts a hydrogen bond to its 13(1)-keto carbonyl. The results show that the Chl binds to a well-defined pocket of the protein and experiences several close contacts with nearby amino acids. From the site-selected fluorescence spectra, it is further concluded that the electron-phonon coupling is moderately strong. Simulations of both the site-selected fluorescence spectra and the temperature dependence of absorption and fluorescence spectra are presented. These simulations indicate that the Huang-Rhys factor characterizing the electron-phonon coupling strength is between 0.6 and 0.9. The width of the Gaussian inhomogeneous distribution function is 210 +/- 10 cm-1.     

FTIR difference spectroscopy in combination with isotope labeling for identification of the carbonyl modes of P700 and P700+ in photosystem I

Room temperature, light induced (P700(+)-P700) Fourier transform infrared (FTIR) difference spectra have been obtained using photosystem I (PS I) particles from Synechocystis sp. PCC 6803 that are unlabeled, uniformly (2)H labeled, and uniformly (15)N labeled. Spectra were also obtained for PS I particles that had been extensively washed and incubated in D(2)O. Previously, we have found that extensive washing and incubation of PS I samples in D(2)O does not alter the (P700(+)-P700) FTIR difference spectrum, even with approximately 50% proton exchange. This indicates that the P700 binding site is inaccessible to solvent water. Upon uniform (2)H labeling of PS I, however, the (P700(+)-P700) FTIR difference spectra are considerably altered. From spectra obtained using PS I particles grown in D(2)O and H(2)O, a ((1)H-(2)H) isotope edited double difference spectrum was constructed, and it is shown that all difference bands associated with ester/keto carbonyl modes of the chlorophylls of P700 and P700(+) downshift 4-5/1-3 cm(-1) upon (2)H labeling, respectively. It is also shown that the ester and keto carbonyl modes of the chlorophylls of P700 need not be heterogeneously distributed in frequency. Finally, we find no evidence for the presence of a cysteine mode in our difference spectra. The spectrum obtained using (2)H labeled PS I particles indicates that a negative difference band at 1698 cm(-1) is associated with at least two species. The observed (15)N and (2)H induced band shifts strongly support the idea that the two species are the 13(1) keto carbonyl modes of both chlorophylls of P700. We also show that a negative difference band at approximately 1639 cm(-1) is somewhat modified in intensity, but unaltered in frequency, upon (2)H labeling. This indicates that this band is not associated with a strongly hydrogen bonded keto carbonyl mode of one of the chlorophylls of P700.     

Chlorophyll a and carotenoid triplet states in light-harvesting complex II of higher plants.

Laser-flash-induced transient absorption measurements were performed on trimeric light-harvesting complex II to study carotenoid (Car) and chlorophyll (Chl) triplet states as a function of temperature. In these complexes efficient transfer of triplets from Chl to Car occurs as a protection mechanism against singlet oxygen formation. It appears that at room temperature all triplets are being transferred from Chl to Car; at lower temperatures (77 K and below) the transfer is less efficient and chlorophyll triplets can be observed. In the presence of oxygen at room temperature the Car triplets are partly quenched by oxygen and two different Car triplet spectral species can be distinguished because of a difference in quenching rate. One of these spectral species is replaced by another one upon cooling to 4 Ki demonstrating that at least three carotenoids are in close contact with chlorophylls. The triplet minus singlet absorption (T-S) spectra show maxima at 504-506 nm and 517-523 nm, respectively. In the Chl Qy region absorption changes can be observed that are caused by Car triplets. The T-S spectra in the Chl region show an interesting temperature dependence which indicates that various Car's are in contact with different Chl a molecules. The results are discussed in terms of the crystal structure of light-harvesting complex II.     

Resonance Raman spectroscopy of horseradish peroxidase derivatives and intermediates with excitation in the near ultraviolet

Resonance Raman enhancement of derivatives and intermediates of horseradish peroxidase in the near ultraviolet (N-band excitation) results in intensity and enhancement patterns that are different from those normally observed within the porphyrin Soret (B-band) and alpha-beta (Q-band) absorptions. In particular it allows the resolution of resonance Raman spectra of horseradish peroxidase compound I. The bands above 1300 cm-1 can be assigned to porphyrin vibrational modes that are characteristically shifted in frequency due to removal of an electron from the porphyrin ring. The resonance Raman frequency shifts follow normal mode compositions. Relative to resonance Raman spectra of compound II, the v4 frequency (primarily Ca-N) exhibits a 20 cm-1 downshift. The v2, v11, and v37 vibrational frequencies whose mode compositions are primarily porphyrin Cb-Cb, exhibit 10-20 cm-1 upshifts. The v3, v10, and v28 frequencies, whose mode compositions are primarily Ca-Cm, exhibit downshifts. The downshifts for v3 and v10 are small, 3-5 cm-1; however, the downshift for v28 is 14 cm-1. These frequency shifts are consistent with those of previously published resonance Raman studies of model compounds. In contrast to reports from other laboratories, the data presented here for horseradish peroxidase compound I can be attributed unambiguously to resonance Raman scattering from a porphyrin pi-cation radical.     

Orientation of emitting dipoles of chlorophyll A in thylakoids: considerations on the orientation factor in vivo.

Orientation angles of five emitting dipoles of chlorophyll a in thylakoids were estimated from low temperature fluorescence polarization ratio spectra of magnetically oriented chloroplasts. A simple expression is given also for the evaluation of data from linear dichroism measurements. It is shown that the Qy dipoles of chlorophylls lie more in the plane of the membranes and span a larger angular interval than was previously thought. Values for the orientation factor are calculated using various models corresponding to different degrees of local order of the Qy dipoles of chlorophylls in the thylakoid. We show that the characteristic orientation pattern of the Qy dipoles of chlorophylls in the membrane, i.e., increasing dichroism toward longer wavelengths, may favour energy transfer between the antenna chlorophylls as well as funnel the excitation energy into the reaction centers.     

The stereospecific interaction between chlorophylls and chlorophyllase. Possible implication for chlorophyll biosynthesis and degradation.

Chlorophyllase-catalyzed hydrolysis and esterification of chlorophylls, bacteriochlorophylls, and their free acids, respectively, depend on the configuration around the C-13(2) atom of the corresponding substrate. The data suggest that the enzyme interacts preferentially with compounds having the isocyclic carbomethoxy and the C-17 propionic residues facing opposite sides of the porphyrin macrocycle. The relevance of this observation to chlorophyll biosynthesis and degradation in vivo is briefly discussed.     

Resonance Raman studies of Rieske-type proteins.

Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferrodoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an St2FeSb2Fe[N(His)]t2 structure for the Rieske-type cluster.     

A resonance Raman study on the structures of complexes of flavoprotein D-amino acid oxidase

Resonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is delta 1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-alpha-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2-symmetric stretching mode of the product, such as delta 1-piperideine-2-carboxylate or delta 1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).     

Reconstitution of the Peridinin–chlorophyll a Protein (PCP): Evidence for Functional Flexibility in Chlorophyll Binding

The coding regions for the N-domain, and full length peridinin–chlorophyll a apoprotein (full length PCP), were expressed in Escherichia coli. The apoproteins formed inclusion bodies from which the peptides could be released by hot buffer. Both the above constructs were reconstituted by addition of a total pigment extract from native PCP. After purification by ion exchange chromatography, the absorbance, fluorescence excitation and CD spectra resembled those of the native PCP. Energy transfer from peridinin to Chl a was restored and a specific fluorescence activity calculated which was ~86% of that of native PCP. Size exclusion analysis and CD spectra showed that the N-domain PCP dimerized on reconstitution. Chl a could be replaced by Chl b, 3-acetyl Chl a, Chl d and Bchl using the N-domain apo protein. The specific fluorescence activity was the same for constructs with Chl a, 3-acetyl Chl a, and Chl d but significantly reduced for those made with Chl b. Reconstitutions with mixtures of chlorophylls were also made with eg Chl b and Chl d and energy transfer from the higher energy Qy band to the lower was demonstrated.     

Structural role of (bacterio)chlorophyll ligated in the energetically unfavorable beta-position

Chlorophyll is attached to apoprotein in diastereotopically distinct ways, by beta- and alpha-ligation. Both the beta- and alpha-ligated chlorophylls of photosystem I are shown to have ample contacts to apoprotein within their proteinaceous binding sites, in particular, at C-13 of the isocyclic ring. The H-bonding patterns for the C-13(1) oxo groups, however, are clearly distinct for the beta-ligated and alpha-ligated chlorophylls. The beta-ligated chlorophylls frequently employ their C-13(1) oxo in H-bonds to neighboring helices and subunits. In contrast, the C-13(1) oxo of alpha-ligated chlorophylls are significantly less involved in H-bonding interactions, particularly to neighboring helices. Remarkably, in the peripheral antenna, light harvesting complex (LH2) from Rhodobacter sphaeroides, a single mutation in the alpha-subunit, introduced to eliminate H-bonding to the beta-bacteriochlorophyll-B850, which is ligated in the "beta-position," results in significant thermal destabilization of the LH2 in the membrane. In addition, in comparison with wild type LH2, the expression level of the LH2 lacking this H-bond is significantly reduced. These findings show that H-bonding to the C-13(1) keto group ofbeta-ligated (bacterio)-chlorophyll is a key structural motif and significantly contributes to the stability of bacteriochlorophyll proteins in the native membrane. Our analysis of photosystem I and II suggests that this hitherto unrecognized motif involving H-bonding to beta-ligated chlorophylls may be equally critical for the stable assembly of the inner core antenna of these multicomponent chlorophyll proteins.     

Probing the primary donor environment in the histidineM200-->leucine and histidineL173-->leucine heterodimer mutants of Rhodobacter capsulatus by light-induced Fourier transform infrared difference spectroscopy.

Light-induced P+QB-/PQB FTIR difference spectra of reaction centers (RCs) have been obtained from chromatophores lacking light-harvesting B800-850 antenna for Rhodobacter capsulatus wild type (WT) and for the two mutants HisM200-->Leu and HisL173-->Leu. The primary donor (P) in both mutants consists of a bacteriochlorophyll-bacteriopheophytin heterodimer. The most prominent difference between the WT and the mutant spectra is in the 1600-1200-cm-1 region. The WT spectrum displays large positive bands at approximately 1290, 1500-1430, and 1580-1530 cm-1. These three bands are either small or altogether absent in the heterodimer spectra. In addition, both heterodimer spectra compare well with the electrochemically generated BChla+/BChla spectrum [M?ntele, W.G., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472]. These observations indicate that the positive charge is localized on the monomeric BChl in the heterodimers. The overall shape of the ester and keto C = O signals in the BChla+/BChla spectrum is maintained in the in situ spectra although significant differences are observed in the frequency, width, and splitting of the bands. The shape of the signal at 1757/1744 cm-1 in HisL173-->Leu is comparable to the 1751/1737-cm-1 signal of BChla+/BChla in tetrahydrofuran, indicating a free 10a ester C = O of PM in HisL173-->Leu. The reduced amplitude of the negative 1740-cm-1 feature in both HisM200-->Leu and WT spectra suggests a hydrogen-bonded 10a ester C = O for PL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

Intramolecular hydrogen bonds and conformation of the lincomycin molecule in organic solvents

IR spectra (1600-1800 and 3000-3650 cm-1) of lincomycin base solutions in inert (CCl4 and C2Cl4), proton acceptor (dioxane, dimethylsulfoxide and triethyl amine) and proton donor (CHCl3, CD3OD and D2O) solvents were studied. Analysis of the concentration and temperature changes in the spectra revealed that association in lincomycin in the inert solvents was due to intramolecular hydrogen linkage involving amide and hydroxyl groups. Disintegration of the associates after the solution dilution and temperature rise was accompanied by formation of intramolecular bonds stabilizing the stable conformation structure of the lincomycin molecule. The following hydrogen linkage in the conformation was realized: NH...N (band v NH...N at 3340 cm-1), OH...O involving the hydroxyl at C-7 and O atoms in the D-galactose ring (band v OH...O at 3548 cm-1), a chain of the hydrogen bonds OH...OH...OH in the lincomycin carbohydrate moiety (band v OH...O at 3593 cm-1 and v OH of the end hydroxyl group at 3625 cm-1). Bonds NH and C-O of the amide group were located in transconformation. Group C-O did not participate in the intramolecular hydrogen linkage.     

Orientational order of cholesteryl oleate in low-density lipoprotein observed by 2H-NMR

Cholesteryl oleate, selectively deuterated at various positions along the acyl chain, has been incorporated into fresh human serum low-density lipoprotein (LDL2). Temperature-dependent 2H-NMR spectra were recorded between 15 and 45 degrees C. For deuterons at C-2' and C-5' of the acyl chain, two 2H-NMR spectral components, a broad and a narrow signal, are observed. This is interpreted as reflecting the coexistence of two cholesteryl ester regions in the LDL2 core which possess different degrees of order. The C-2H bond order parameters, SCD, are approx. 0.12-0.20 for the more ordered region and approx. 0.04-0.06 for the less ordered region. Longitudinal relaxation times, T1, of deuterated cholesteryl oleate are found to increase between C-8' and the terminal -C2H3 group, which is consistent with an increased rate of chain motion toward the free ends of the ester acyl chains.     

Resonance Raman characterization of Rhodobacter sphaeroides reaction centers bearing site-directed mutations at tyrosine M210

Resonance Raman (RR) spectroscopy and low-temperature absorption spectroscopy have been used to investigate the structural changes in the reaction centers (RCs) of Rhodobacter sphaeroides induced by site-specific mutations on the tyrosine (Y) M210 residue. RCs in which Y M210 has been genetically replaced with phenylalanine (F) or leucine (L) exhibit a 5-fold decrease in their primary electron-transfer kinetics (Finkele et al., 1990). The general similarity of RR spectra of the wild-type RCs as compared to those of the two mutant RCs indicates that no significant global structural changes occur upon mutation at the level of any of the six bacteriochlorin pigments. In the RR spectra of the two mutant RCs there is a conspicuous absence of contributions from the BPheM prosthetic group, which is interpreted in terms of a change in the resonance enhancement conditions of this chromophore. Low-temperature adsorption spectroscopy reveals marked shifts in the Qx absorption band of BPheM. This shift is interpreted as arising from a destabilization of the protein in the vicinity of BPheM and accounts for the change in resonance condition for this chromophore in its RR contributions. As well, there is a 3-nm red shift of the Qy absorption band of the BChls from 803 to 806 nm for the mutant RCs. Difference RR spectra yielding structural information concerning, selectively, the primary donor (P) indicate that the structure of the P binding pocket is conserved for these mutant RCs. The tyrosine M210 is not observed to be engaged in a hydrogen bond with either of the acetyl or keto carbonyls of P.