The influence of hydrogen bonds on the electronic structure of light-harvesting complexes from photosynthetic bacteria.

The influence of hydrogen bonds on the electronic structure of the light-harvesting I complex from Rhodobacter sphaeroides has been examined by site-directed mutagenesis, steady-state optical spectroscopy, and Fourier-transform resonance Raman spectroscopy. Shifts of 4-23 nm in the Q(y) absorption band were observed in seven mutants with single or double changes at Leu alpha44, Trp alpha43, and Trp beta48. Resonance Raman spectra were consistent with the loss of a hydrogen bond with the alteration of either Trp alpha43 or Trp beta48 to Phe. However, when the Trp alpha43 to Phe alteration is combined with Leu alpha44 to Tyr, the spectra show that the loss of the hydrogen bond to alpha43 is compensated by the addition of a new hydrogen bond to Tyr alpha44. Comparison of the absorption and vibrational spectra of the seven mutants suggests that changes in the absorption spectra can be interpreted as being due to both structural and hydrogen-bonding changes. To model these changes, the structural and hydrogen bond changes are considered to be independent of each other. The calculated shifts agree within 1 nm of the observed values. Excellent agreement is also found assuming that the structural changes arise from rotations of the C3-acetyl group conformation and hydrogen bonding. These results provide the basis for a simple model that describes the effect of hydrogen bonds on the electronic structures of the wild-type and mutant light-harvesting I complexes and also is applicable for the light-harvesting II and light-harvesting III complexes. Other possible effects of the mutations, such as changes in the disorder of the environment of the bacteriochlorophylls, are discussed.

Functional implications of the propionate 7-arginine 220 interaction in the FixLH oxygen sensor from Bradyrhizobium japonicum.

BjFixL from Bradyrhizobium japonicum is a heme-based oxygen sensor implicated in the signaling cascade that enables the bacterium to adapt to fluctuating oxygen levels. Signal transduction is initiated by the binding of O(2) to the heme domain of BjFixL, resulting in protein conformational changes that are transmitted to a histidine kinase domain. We report structural changes of the heme and its binding pocket in the Fe(II) deoxy and Fe(III) met states of the wild-type BjFixLH oxygen sensor domain and four mutants of the highly conserved residue arginine 220. UV-visible, electron paramagnetic resonance, and resonance Raman spectroscopies all showed that the heme iron of the R220H mutant is unexpectedly six-coordinated at physiological pH in the Fe(III) state but undergoes pH- and redox-dependent coordination changes. This behavior is unprecedented for FixL proteins, but is reminiscent of another oxygen sensor from E. coli, EcDos. All mutants in their deoxy states are five-coordinated Fe(II), although we report rupture of the residue 220-propionate 7 interaction and structural modifications of the heme conformation as well as propionate geometry and flexibility. In this work, we conclude that part of the structural reorganization usually attributed to O(2) binding in the wild-type protein is in fact due to rupture of the Arg220-P7 interaction. Moreover, we correlate the structural modifications of the deoxy Fe(II) states with k(on) values and conclude that the Arg220-P7 interaction is responsible for the lower O(2) and CO k(on) values reported for the wild-type protein.

Calcium binding to the photosystem II subunit CP29.

We have identified a Ca(2+)-binding site of the 29-kDa chlorophyll a/b-binding protein CP29, a light harvesting protein of photosystem II most likely involved in photoregulation. (45)Ca(2+) binding studies and dot blot analyses of CP29 demonstrate that CP29 is a Ca(2+)-binding protein. The primary sequence of CP29 does not exhibit an obvious Ca(2+)-binding site therefore we have used Yb(3+) replacement to analyze this site. Near-infrared Yb(3+) vibronic side band fluorescence spectroscopy (Roselli, C., Boussac, A., and Mattioli, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12897-12901) of Yb(3+)-reconstituted CP29 indicated a single population of Yb(3+)-binding sites rich in carboxylic acids, characteristic of Ca(2+)-binding sites. A structural model of CP29 presents two purported extra-membranar loops which are relatively rich in carboxylic acids, one on the stromae side and one on the lumenal side. The loop on the lumenal side is adjacent to glutamic acid 166 in helix C of CP29, which is known to be the binding site for dicyclohexylcarbodiimide (Pesaresi, P., Sandonà, D., Giuffra, E. , and Bassi, R. (1997) FEBS Lett. 402, 151-156). Dicyclohexylcarbodiimide binding prevented Ca(2+) binding, therefore we propose that the Ca(2+) in CP29 is bound in the domain including the lumenal loop between helices B and C.

Conformation of bacteriochlorophyll molecules in photosynthetic proteins from purple bacteria.

Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [Näveke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.

Surface-enhanced Raman scattering and fluorescence spectroscopy reveal molecular interactions of all-trans retinoic acid and RAR gamma ligand-binding domain.

Surface-enhanced Raman scattering and fluorescence were used to investigate the interactions of all-trans retinoic acid with the gamma-type retinoic acid receptor. Raman data revealed a significant attenuation in intensity of the bands originating from the retinoic acid polyenic chain upon receptor binding, with the spectrum being dominantly that of the beta-ionone ring. Fluorescence measurements supported the hydrophobic character of the ligand binding. These novel spectroscopic results are fully consistent with the published X-ray crystallographic data and suggest that these techniques may be valuable additional tools to characterize the interactions of agonists and antagonists with residues in the ligand-binding pockets of retinoid receptor homo- and heterodimers.

Effects of hydrogen bonds on the redox potential and electronic structure of the bacterial primary electron donor.

The primary donor, P, of photosynthetic bacterial reaction centers (RCs) is a dimer of excitonically interacting bacteriochlorophyll (BChl) molecules. The two constituents are named PL and PM to designate their close association with the L- and M-subunits, respectively, of the RC protein. A series of site-directed mutants of RCs from Rhodobacter sphaeroides has been constructed in order to model the effects of hydrogen bonding on the redox midpoint potential and electronic structure of P. The leucine residue at position M160 was genetically replaced with eight other amino acid residues capable of donating a hydrogen bond to the C9 keto carbonyl group of the PM BChl a molecule of P. Fourier transform (FT) (pre)resonance Raman spectroscopy with 1064 nm excitation was used to (i) determine the formation and strengths of hydrogen bonds on this latter keto carbonyl group in the reduced, neutral state (PO), and (ii) determine the degree of localization of the positive charge on one of the two constituent BChl molecules of P in its oxidized, radical cation state (P*+). A correlation was observed between the strength of the hydrogen bond and the increase in PO/P*+ redox midpoint potential. This correlation is less pronounced than that observed for another series of RC mutants where hydrogen bonds to the four pi-conjugated carbonyl groups of P were broken or formed uniquely involving histidinyl residues [Mattioli, T. A., Lin, X., Allen, J. P. and Williams, J. C. (1995) Biochemistry 34, 6142-6152], indicating that histidinyl residues are more effective in raising the PO/P*+ redox midpoint potential via hydrogen bond formation than are other hydrogen bond-forming residues. In addition, an increase in positive charge localization is correlated with the strength of the hydrogen bond and with the PO/P*+ redox midpoint potential. This latter correlation was analyzed using an asymmetric bacteriochlorophyll dimer model based on Hückel-type molecular orbitals in order to obtain estimates of certain energetic parameters of the primary donor. Based on this model, the correlation is extrapolated to the case of complete localization of the positive charge on PL and gives a predicted value for the P/P+ redox midpoint potential similar to that experimentally determined for the Rb. sphaeroides HL(M202) heterodimer. The model yields parameters for the highest occupied molecular orbital energies of the two BChl a constituents of P which are typical for the oxidation potential of isolated BChl a in vitro, suggesting that the protein, as compared to many solvents, does not impart atypical redox properties to the BChl a constituents of P.

Temperature-dependent behavior of bacteriochlorophyll and bacteriopheophytin in the photosynthetic reaction center from Rhodobacter sphaeroides.

We have reexamined the temperature dependence of resonance Raman (RR) spectra of the bacteriochlorin cofactors bound to reaction centers from Rhodobacter sphaeroides. Three types of resonant excitations were performed, namely, Soret band, bacteriopheophytin Qx-band, and near-infrared, Qy-band (pre)resonances. Sample temperature was varied from 300 to 10 K. In both Soret-resonant and Qy-preresonant Raman spectra, the ca. 1610-cm(-1) band corresponding to a bacteriochlorophyll CaCm methine bridge stretching mode is observed to increase in frequency by 4-6 cm(-1) as temperature is decreased from 300 to 15 K. This upshift is interpreted as arising from a change in conformation of the bacteriochlorophyll macrocycles. It may be nonspecific to the protein-bound cofactors, since a similar 4-cm(-1) upshift was observed in the same temperature range for BChl a in solution. Qx-resonant Raman spectra of either of the two bacteriopheophytin (BPhe) cofactors were obtained selectively using excitations at 537 and 546 nm. No significant frequency shift was observed for the CaCm stretching mode of BPheL between 200 and 15 K. We conclude, at variance with a previous report, that the macrocycle of the BPheL primary electron acceptor does not undergo any significant conformational change in the 200-15 K temperature range. Qy-preresonant excitation of RCs at 1064 nm provided selective Raman information on the primary electron donor (P primary). The stretching frequencies of the two conjugated keto and acetyl carbonyl groups of the M-branch primary donor BChl cofactor (P(M)) did not significantly change between 300 and 10 K. In contrast the keto carbonyl stretching frequency of cofactor P(L) was observed to upshift by 5 cm(-1), while its acetyl carbonyl frequency downshifted by 2 cm(-1). The latter shift indicated that the strong H-bond between the acetyl group of P(L) and His L168 may have slightly strengthened at 10 K. Excitation at 1064 nm of chemically oxidized RCs selectively provided RR spectra of the primary donor in its radical P.+ state. These spectra can be interpreted as a decrease of the localization of the positive charge on P(L) from 78% to 63% when the temperature decreased from 300 to 10 K resulting in a more electronically symmetric dimer. Possible origins of the temperature dependence of the positive charge delocalization in P.+ are discussed.

Influence of Asn/His L166 on the hydrogen-bonding pattern and redox potential of the primary donor of purple bacterial reaction centers.

The primary electron donor (P) of the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter (Rb.) sphaeroides is constituted of two bacteriochlorophyll molecules in excitonic interaction. The C2 acetyl carbonyl group of one of the two bacteriochlorophyll molecules (PL), the one more closely associated with the L polypeptide subunit, is engaged in a hydrogen bond with histidine L168, while the other pi-conjugated carbonyl groups of P are free from such hydrogen-bonding interactions. The three-dimensional X-ray crystal structures of the RC from several strains of Rb. sphaeroides reveal that asparagine L166 probably interacts indirectly with P through His L168. Such an interaction is expected to modulate the hydrogen bond between P and His L168, a residue which is highly conserved in purple bacteria. RC mutants of Rb. sphaeroides where asparagine L166 was genetically replaced by leucine [NL(L166)], histidine [NH(L166)], and aspartate [ND(L166)] were studied using Fourier transform (FT) Raman spectroscopy. All of these mutations resulted in an increase in the strength of the hydrogen bond between His L168 and the acetyl carbonyl group of P(L), as observed in the FT Raman spectrum, by the 2-4 cm(-1) decrease in vibrational frequency of the 1620 cm(-1) band which has been assigned to this specific acetyl carbonyl group [Mattioli, T. A., Lin, X., Allen, J. P., & Williams, J. C. (1995) Biochemistry 34, 6142-6152]. At pH 8, the NH(L166) mutation showed the greatest change in the P0/P.+ redox midpoint potential (515 mV), increasing it by ca. 30 mV compared to that of wild type (485 mV). A similar increase in P0/P.+ redox midpoint potential for NH(L166) compared to that of wild type is also observed at pH 5, 6, and 9.5. The p0/P.+ midpoint potential of the NL(L166) mutant was comparable to that of wild type at all pH values. In contrast, for the ND(L166) mutant, the midpoint potential shows a markedly different pH dependency, being 25 mV higher than wild type at pH 5 but 20 mV lower than wild type at pH 9.5. The hydrogen bond interactions of the primary electron donor from Rhodospirillum (Rsp.) centenum were determined from the FT Raman vibrational spectrum which exhibits a 1616 cm(-1) band similar to what is seen in the NH(L166) and ND(L166) Rb. sphaeroides mutants. Comparison of the sequence of the L subunit determined for the Rsp. centenum RC with that of other species indicates that positions L166 and L168 are occupied by His residues. The stronger hydrogen bond between the conserved His L168 and the acetyl carbonyl group of P(L), observed in the primary donor of Rsp. centenum and of several bacterial species which are known to possess a histidine residue at the analogous L166 position, is proposed to be due to interactions between these two histidine residues.

Site-directed mutations near the L-subunit D-helix of the purple bacterial reaction center: a partial model for the primary donor of photosystem II.

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem II (PS II) reaction center (RC). To construct this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized, such that an 11-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which constitutes the primary donor are substantially altered by these modifications, to the degree that the dimer becomes functionally much more "monomeric". The changes include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P, from /D/ = 185 x 10(-4) cm(-1) and /E/ = 31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/ = 200 x 10(-4) cm(-1) and /E/ = 44 x 10(-4) cm(-1) in the mutant and (2) an increase in the EPR line width of the oxidized state, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of 3P in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potential in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl on the active electron transfer pathway (P(A)) is absent in the mutant, due to the fact that HisL168 in the WT sequence has been replaced by a leucine in D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C9 keto carbonyl oxygen of P(A) and an unknown hydrogen-bond donor. Thus, although the protein environment around one of the Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary donor that is oriented like the one in PS II.

Detection of a Yb3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups.

Near infrared Yb3+ vibronic sideband spectroscopy was used to characterize specific lanthanide binding sites in bacteriorhodopsin (bR) and retinal free bacteriorhodopsin (bO). The VSB spectra for deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are identical. Application of a two-dimensional anti-correlation technique suggests that only a single Yb3+ site is observed. The Yb3+ binding site in bO is observed to consist of PO2- groups and carboxylic acid groups, both of which are bound in a bidentate manner. An additional contribution most likely arising from a phenolic group is also observed. This implies that the ligands for the observed single binding site are the lipid head groups and amino acid residues. The vibronic sidebands of Yb3+ in deionized bR regenerated at a ratio of 2:1 ion to bR are essentially identical to those in bO. The other high-affinity binding site is thus either not evident or its fluorescence is quenched. A discussion is given on the difference in binding of Ca2+ (or Mg2+) and lanthanides in phospholipid membrane proteins.

Hydrogen-bond interactions of the primary donor of the photosynthetic purple sulfur bacterium Chromatium tepidum.

We have used near-infrared Fourier transform (pre)resonance Raman spectroscopy to determine the protein interactions with the bacteriochlorophyll (BChl) dimer constituting the primary electron donor, P, in the reaction center (RC) from the thermophilic purple sulfur bacterium Chromatium tepidum. In addition, we report the alignment of partial sequences of the L and M protein subunits of C. tepidum RCs in the vicinity of the primary donor with those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. Taken together, these results enable us to propose the hydrogen-bonding pattern and the H-bond donors to the conjugated carbonyl groups of P. Selective excitation (1064-nm laser radiation) of the FT (pre)-resonance Raman spectra of P in its neutral (P degree) and oxidized (P degree +) states were obtained via their electronic absorption bands at 876 and 1240 nm, respectively. The P degree spectrum exhibits vibrational frequencies at 1608, 1616, 1633, and 1697 cm-1 which bleach upon P oxidation. The P degree + spectrum exhibits new bands at 1600, 1639, and 1719 cm-1. The 1608-cm-1 band, which downshifts to 1600 cm-1 upon oxidation, is assigned to a CaCm methine bridge stretching mode of the P dimer, indicating that each BChl molecule possesses a single axial ligand (His L181 and His M201, from the sequence alignment). The 1616- and 1633-cm-1 bands correspond to two H-bonded pi-conjugated acetyl carbonyl groups of each BChl molecule. with different H-bond strengths: the 1616-cm-1 band is assigned to the PL C2 acetyl group which is H-bonded to a histidine residue (His L176), while the 1633-cm-1 band is assigned to the PM C2 acetyl carbonyl, H-bonded to a tyrosine residue (Tyr M196). Both PL and PM C9 keto carbonyls are free from interactions and vibrate at the same frequency (1697 cm-1). Thus, the H-bond pattern of the primary donor of C. tepidum differs from that of Rb. sphaeroides in the extra H-bond to the PM C2 acetyl carbonyl group; that of PL is H-bonded to a histidine residue in both primary donors (His L168 in Rb. sphaeroides and His L176 in C. tepidum). The P degree/P degree + redox midpoint potentials were measured to be +497 and +526 mV for isolated C. tepidum RCs with and without the associated tetraheme cytochrome c subunit, respectively, and +502 mV for intracytoplasmic membranes. The positive charge localization was estimated to be 69% in favor of PL, indicating a more delocalized situation over the primary donor of C. tepidum than that of Rb. sphaeroides (estimated to be 80% on PL). These differences in physicochemical properties are discussed with respect to the proposed structural model for the microenvironment of the primary donor of C. tepidum.

Effects of hydrogen bonding to a bacteriochlorophyll-bacteriopheophytin dimer in reaction centers from Rhodobacter sphaeroides.

The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)--bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations: His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn M., Lin, X., Williams, J. C., Allen J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for the addition of a hydrogen bond to the 9-keto and 2-acetyl carbonyl groups. The oxidation--reduction midpoint potential of the donor is increased by approximately 85 mV by the addition of a hydrogen bond to the BChl molecule but is only increased by approximately 15 mV by the addition of a hydrogen bond to the BPhe molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potential. The yield of electron transfer to the primary quinone is 5-fold reduced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the donor Qy band in the mutant that adds a hydrogen bond to the BChl molecule. The changes in the optical spectra and the yield of electron transfer are consistent with a model in which the addition of a hydrogen bond to the BChl molecule increases the energy of an internal charge transfer state while the addition to the BPhe molecule stabilizes this state. The results show that the properties of the heterodimer are different depending on which side is hydrogen-bonded and suggest that the hydrogen bonds alter the energy of the internal charge transfer state in a well-defined manner.

Structure and protein binding interactions of the primary donor of the Chloroflexus aurantiacus reaction center.

Soret resonance, QX resonance, and QY near-infrared Fourier transform (FT) (pre)resonance Raman spectroscopies were used to determine pigment-protein interactions of specific bacteriochlorin molecules in the reaction center from Chloroflexus aurantiacus. FT Raman spectroscopy, using 1064 nm excitation, was used to selectively obtain preresonance and resonance vibrational Raman spectra of the primary donor (P) of reaction centers (RCs) from Chloroflexus aurantiacus in the Po and P.+ states, respectively. The FT Raman spectrum of RCs in their neutral P (Po) state exhibits bands at 1605, 1632, 1648, and 1696 cm-1 which are attributable to P in its resting neutral state. Specifically, the latter three Raman bands can be assigned to the conjugated C2 acetyl and C9 keto carbonyl groups of the bacteriochlorophyll (BChl) molecules constituting P. The observation of at least three such bands is indicative of a non-monomeric nature of P, consistent with the proposal that it is a dimer of BChl molecules. The 1632 cm-1 band is consistent only with a hydrogen bonded BChl acetyl carbonyl, while the 1648 cm-1 band is assigned to a non-hydrogen bonded acetyl carbonyl. The 1696 cm-1 band is consistent only with a non-hydrogen bonded keto carbonyl group; from the unusually high intensity of this latter band compared to the others, we propose that the 1696 cm-1 band contains contributions from two keto carbonyl groups, both free of hydrogen bonds. From published protein sequence alignments of the L and M subunits of Rhodobacter (Rb.) sphaeroides and Chloroflexus aurantiacus we assign the 1632 cm-1 band as arising from the C2 acetyl carbonyl of the analogous PM constituent of P, which is hydrogen bonded to tyrosine M187 in the Chloroflexus RC, and propose a pigment-protein structural model for the primary donor of Chloroflexus aurantiacus. The FT Raman spectrum of RCs in the P degrees+ state indicates that one component of the 1696 cm-1 band has upshifted 21 cm-1 to 1717 cm-1. Compared to Rb. sphaeroides which showed a 26 cm-1 upshift for the corresponding band, the 21 cm-1 upshift indicates that the + charge is more delocalized over the P.+ species of Chloroflexus; we estimate that ca. 65% of the + charge is localized on one of the two BChl molecules of the Chloroflexus primary donor as compared to ca. 80% for Rb. sphaeroides. The consequences of the proposed structure of the Chloroflexus primary donor in terms of its Po/P.+ redox midpoint potential are discussed.

Time-resolved and steady-state spectroscopic analysis of membrane-bound reaction centers from Rhodobacter sphaeroides: comparisons with detergent-solubilized complexes.

The spectroscopic analysis of the antenna-deficient Rhodobacter sphaeroides strain RCO1 has been extended to an investigation of the kinetics and spectroscopy of primary charge separation. Global analysis of time-resolved difference spectra demonstrated that the rate of charge separation in membrane-bound reaction centers is slightly slower than in detergent-solubilized reaction centers from the same strain. A kinetic analysis of the decay of the primary donor excited state at single wavelengths was carried out using a high repetition rate laser system, with the reaction centers being maintained in the open state using a combination of phenazine methosulfate and horse heart cytochrome c. The kinetics of primary charge separation in both membrane-bound and solubilized reaction centers were found to be non-monoexponential, with two exponential decay components required for a satisfactory description of the decay of the primary donor excited state. The overall rate of charge separation in membrane-bound reaction centers was slowed if the primary acceptor quinone was reduced using sodium ascorbate. This slowing was caused, in part, by an increase in the relative amplitude of the slower of the two exponential components. The acceleration in the rate of charge separation observed on removal of the reaction center from the membrane did not appear to be caused by a significant change in the electrochemical properties of the primary donor. The influence of the environment of the reaction center on primary charge separation is discussed together with the origins of the non-monoexponential decay of the primary donor excited state.

Symmetric structural features and binding site of the primary electron donor in the reaction center of Chlorobium.

The protein binding interactions of the constituent bacteriochlorophyll a molecules of the primary electron donor, P840, in isolated reaction centers from Chlorobium limicola f thiosulphatophilum and the electronic symmetry of the radical cation P840+. were determined using near-infrared Fourier transform (FT) Raman spectroscopy excited at 1064 nm. The FT Raman vibrational spectrum of P840 indicates that it is constituted of a single population of BChl a molecules which are spectrally indistinguishable. The BChl a molecules of P840 are pentacoordinated with only one axial ligand on the central Mg atom, and the pi-conjugated C2 acetyl and C9 keto carbonyls are free of hydrogen-bonding interactions. The FT Raman spectrum of P840+. exhibits a 1707 cm-1 band attributable to a BChl a C9 keto carbonyl group vibrational frequency that has upshifted 16 cm-1 upon oxidation of P840; this upshift is exactly one-half of that expected for the one-electron oxidation of monomeric BChl a in vitro. The 16 cm-1 upshift, thus, indicates that the resulting +1 charge is equally shared between two BChl a molecules. This situation is markedly different from that of the oxidized primary donor of the purple bacterial reaction center of Rhodobacter sphaeroides, (i) which exhibits a 1717 cm-1 band that has upshifted 26 cm-1, indicating an asymmetric distribution of the resulting +1 charge over the two constituent BChl a molecules, and (ii) whose H-bonding pattern with respect to the pi-conjugated carbonyl groups is asymmetric.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between multiple hydrogen bonding and alteration of the oxidation potential of the bacteriochlorophyll dimer of reaction centers from Rhodobacter sphaeroides.

The electronic absorption and vibrational Raman spectra of mutant reaction centers from Rhodobacter sphaeroides bearing multiple site-specific mutations near the primary electron donor (P), a bacteriochlorophyll dimer, are reported. These mutations bear double and triple combinations of single-point mutations that alter the H-bonding interactions between histidine residues and the C2- and C9-conjugated carbonyl groups of the primary donor [Mattioli, T.A., Williams, J.C., Allen, J.P., & Robert, B. (1994) Biochemistry 33, 1636-1643] and change the donor redox midpoint potential from 410 to 765 mV compared to 505 mV for wild type [Lin, X., Murchison, H.A., Nagarajan, V., Parson, W.W., Williams, J.C., & Allen, J.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269]. Near-infrared Fourier transform Raman spectroscopy was used to determine the changes in H-bonding interactions of the primary donor in these multiple mutants. The Fourier transform Raman spectra of the mutants exhibit the predicted changes in hydrogen bond interactions of the P carbonyl groups with the protein, and they are consistent with the designed mutations. Moreover, the Raman data verify that the H-bonds formed or broken in the multiple mutants are similar in strength to those observed in the corresponding single mutants. A correlation was observed between the change in P/P.+ redox midpoint potential and the total change in H-bonding interaction energy (from -207 to 364 meV relative to wild type) as gauged by the estimated enthalpy of each H-bond formed or broken on the four conjugated carbonyls of the primary donor. Only minor changes were observed in the optical spectra of the mutant reaction centers, indicating that the addition of H-bonds from histidines has little effect in destabilizing the first electronic excited state of the dimer relative to the ground state. However a blue shift in the dimer absorption band at ca. 890 nm at 20 K was associated with the removal of the H-bond to the C2 acetyl carbonyl group via His L168. A red shift of the oxidized dimer band at ca. 1250 nm was associated with the formation of each H-bond to the C9 keto carbonyl groups.

Direct vibrational structure of protein metal-binding sites from near-infrared Yb3+ vibronic side band spectroscopy.

Near-infrared Yb3+ vibronic side band (VSB) spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This technique provides a selective "IR-like" vibrational spectrum of those ligands chelated to the Yb3+ ion. VSB spectra of various model complexes of Yb3+ representing different ligand types were studied to provide references for the VSB spectra of Yb(3+)-reconstituted metalloproteins. Ca2+ in the calcium-binding protein parvalbumin and Fe3+ in the iron-transporting protein transferrin were replaced with Yb3+. The fluorescence of Yb3+ reconstituted into these two proteins exhibits weak VSBs whose energy shifts, with respect to the main 2F5/2-->2F7/2 Yb3+ electronic transition, represent the vibrational frequencies of the Yb3+ ligands. The chemical nature of the ligands of the Yb3+ in these proteins, as deduced by the observed VSB frequencies, is entirely in agreement with their known crystal structures. For transferrin, replacement of the 12CO3(2-) metal counterion with 13CO3(2-) yielded the expected isotopic shift for the VSBs corresponding to the carbonate vibrational modes. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.

Structure and binding site of the primary electron acceptor in the reaction center of Chlorobium.

In isolated, chlorosome-free reaction centers from Chlorobium limicola f thiosulphatophilum, a chlorin pigment exhibits a Qy absorption band at 672 nm (Feiler, U., Nitschke, W., & Michel, H. (1992) Biochemistry 31, 2608-2614). To characterize the chemical nature of this chlorin pigment and its interactions within the reaction-center protein, selective enhancement of its Raman scattering was achieved by resonant excitation within its Soret band. This is the first time that structural studies of this pigment were performed on the native reaction-center protein. The obtained resonance Raman spectra were consistent with a single population of a chlorophyll a(-like) pigment, possessing a vinyl group on ring I, but not with bacteriochlorophyll c or bacteriophaeophytin c. The stretching frequencies of the C9-keto carbonyl of this pigment indicates that it is H-bonded to the reaction-center protein. The strength of this H-bond is very close to those of the keto carbonyls of the primary electron acceptors in purple bacterial reaction centers and D1/D2 particles. Since in membranes of Chlorobiaceae a transient bleaching at 670 nm is due to the primary acceptor in the reaction center (Nuijs, A. M., Vasmel, H., Joppe, H. L. P., Duysens, L. N. M., & Amesz, J. (1985a) Biochim. Biophys. Acta 907, 24-34), we thus conclude that the primary acceptor in Chlorobium reaction centers is the characterized chlorophyll a(-like) pigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in primary donor hydrogen-bonding interactions in mutant reaction centers from Rhodobacter sphaeroides: identification of the vibrational frequencies of all the conjugated carbonyl groups.

Specific changes in the hydrogen-bonding states of the primary donor, P, in reaction centers from Rhodobacter sphaeroides bearing mutations near P were determined using near-infrared excited Fourier transform (FT) Raman spectroscopy. This technique, using 1064-nm excitation, provides the preresonantly enhanced vibrational spectrum of P in its reduced state selectively over the contributions of the other reaction center chromophores and protein and yields structural information concerning P and its hydrogen-bonding interactions. The mutations studied were as follows: Leu M160-->His, Leu L131-->His, the D9 double mutant (Leu M160-->His + Leu L131-->His), Phe M197-->His, and His L168-->Phe. These mutations were designed to introduce new, or to break existing, hydrogen bonds to the C9 and C2 carbonyl groups of P. On the basis of previous assignments [Mattioli, T. A., Hoffmann, A., Robert, B., Schrader, B., & Lutz, M. (1991) Biochemistry 30, 4648-4654], the FT Raman spectra of these mutants show the predicted changes in hydrogen bond interactions of P carbonyl groups with the protein. The results of this study have permitted us to unambiguously identify the C2 and C9 carbonyl vibrators of P in Rb. sphaeroides. The genetically introduced hydrogen bond interactions are discussed in terms of other physicochemical properties of P including the redox potential and electronic asymmetry in the P+ state. It is discussed that changes in protein hydrogen bonding to the conjugated carbonyl groups of P alone are not the sole factor that contributes to the sizeable modifications of the P/P+ redox midpoint potentials, and that the chemical nature of the hydrogen bond donor plays a significant role in this modification.

Site-specific mutagenesis of the reaction centre from Rhodobacter sphaeroides studied by Fourier transform Raman spectroscopy: mutations at tyrosine M210 do not affect the electronic structure of the primary donor.

The effects of mutation of residue tyrosine M210 on the primary donor bacteriochlorophylls have been investigated by near infrared FT-Raman spectroscopy in reaction centres purified from an antenna-deficient strain of Rhodobacter sphaeroides. We find that mutation at the M210 position does not significantly perturb the distribution of the unpaired electron over the pair of bacteriochlorophyll molecules which constitute the primary donor radical cation. We conclude, therefore, that the effects of mutation of tyrosine M210 on the rate and asymmetry of primary electron transfer in reaction centres cannot be ascribed to a change in the electronic structure of the primary donor.