Gérard Berger

The stereoisomerism of bacterial, reaction-center-bound carotenoids revisited: An electronic absorption, resonance Raman and 1H-NMR study

Abstract In order to solve discrepancies between earlier assignments we have reinvestigated the stereoisomerism of the spheroidene molecule bound to reaction centers (RC) of Rhodobacter sphaeroides. A stable cis isomer could be extracted and purified from the reaction centres by working at very low ambient light. Resonance Raman spectroscopy showed that this cis isomer assumed the same configuration as that of the RC-bound molecule. Proton-NMR spectroscopy of the extracted isomer permitted to assign it the 15–15′ mono cis configuration. Comparisons between resonance Raman spectra of the native form and of the 15 cis extract showed that, in the reaction center, 15 cis spheroidene is in addition twisted into a non-planar conformation. Comparisons of extraction-induced changes in relative intensities of Raman bands of the 760–1060 cm−1 regions, which largely correspond to out-of-plane modes, further indicated that the out-of-plane twist of RC-bound spheroidene should predominantly affect C8–C12 and/or C8′–C12′ regions of the molecule rather than the central region. Comparisons between difference electronic absorption spectra of RC-bound spheroidene and of RC-bound methoxyneurosporene showed that the out-of-plane twisting of both these native forms results in a drastic weakening of their 1C ← 1A electronic transitions, compared with those of the planar, 15 cis forms. Finally, it is proposed, on the basis of their resonance Raman spectra, that spirilloxanthin bound to RCs of Rhodospirillum rubrum as well as dihydroneurosporene or dihydrolycopene bound to RCs of Rhodopseudomonas viridis shares 15 cis configurations and out-of-plane twisting with carotenoids bound to RCs of various strains of Rb. sphaeroides.

CHARACTERIZATION OF BONDING INTERACTIONS OF THE INTERMEDIARY ELECTRON ACCEPTOR IN THE REACTION CENTER OF PHOTOSYSTEM II BY FTIR SPECTROSCOPY

Abstract Molecular changes associated with the photoreduction of the pheophytin a intermediary electron acceptor in films of Photosystem II reaction center (D1D2 RC) were characterized by FTIR spectroscopy. Upon accumulation at 240 K of the photoreduced acceptor, three negative carbonyl bands are observed at 1739 cm−1, 1721 cm−1 and 1677 cm−1 in the light-minus-dark FTIR spectrum of D1D2 RC. The redox-induced FTIR spectrum of the pheophytin a anion generated electrochemically in tetrahydrofuran shows only two negative bands at 1743 cm−1 and 1706 cm−1 which are assigned to changes of absorption of the 10a-ester C=O and 9-keto C=O, respectively. These assignments are based upon the comparison between FTIR data obtained on radicals of pheophytin a and its pyroderivative lacking the 10a-ester C=O. Thus, the 1677 cm−1 band observed in vivo reflects an interacting 9-keto C=O in D1D2 RC. The close similarity observed between: (i) FTIR spectra obtained on Photosystem II and Rps. viridis reaction centers and (ii) amino-acid sequences of the L and D1 polypeptides leads to the assignment of the 1721 cm−1 band in D1D2 RC to a protein-bound 10a-ester C=O of the acceptor and the 1739 cm−1 band to a contribution from the protonated carboxylic group of Glu D1-130 which is proposed to be H-bonded to the 9-keto C=O of the pheophytin acceptor, in the same way as in the Rps. viridis reaction center, Glu L104 is interacting with the 9-keto C=O of HL. The FTIR data indicate that the interactions of the 9-keto C=O and of the 10a-ester of the intermediary acceptor with the protein are stronger in D1D2 RC than in Rps. viridis. These stronger interactions could account, at least in part, for the difference in accessibility to 1H-2H exchange of the H-bonded proton of the Glu D1-130 side-chain in D1D2 RC compared to Rps. viridis reaction center.

Comparison of α-helix orientation in the chromatophore, quantasome and reaction centre of Rhodopseudomonas viridis by circular dichroism and polarized infrared spectroscopy

A comparison of the protein structure in chromatophores, quantasomes and reaction centres of Rhodopseudomonas viridis is made by investigating ultraviolet circular dichroism and polarized infrared spectra of the intact chromatophore membrane, the isolated quantasome and the reaction centre reconstituted into phosphatidyl choline vesicles. A quantasome (photoreceptor unit) is made of a reaction centre surrounded by a ring of antenna complexes. Conformational analysis of the circular dichroism data indicates that intact chromatophores and quantasomes contain more α-helical structure (57%) than reaction centres (47%). Infrared dichroism data show that α-helical segments are on the average closer to the membrane normal in quantasomes (фα = 28°) than in reaction centres (фα = 38°C). This suggests a higher content of α-helices and a better orientation of transmembrane α-helical rods in the antennae surrounding the reaction centre. Our data are discussed in view of the results previously obtained by infrared dichroism of reaction centre films of Rps. sphaeroides (Nabedryk, E., Tiede, D.M., Dutton, P.L. and Breton, J. (1984) in Advances in Photosynthesis Research (Sybesma, C., ed.), Vol. II, pp. 177–180, Martinus Nijhoff/Dr. W. Junk Publishers, Dordrecht, The Netherlands) and by X-rays of reaction centre crystals of Rps. viridis (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398). Our results also imply that the secondary structure and orientation of the protein is comparable in bacteriochlorophyll a- and bacteriochlorophyll b-containing reaction centres. Furthermore, our data on the orientation of the α-helices in the reaction centre of Rps. viridis imply that the C-2 symmetry axis observed in the model derived from X-ray crystallography is oriented in vivo along the normal to the membrane plane.

KINETICS OF ATP HYDROLYSIS BY THE F1-ATPASE FROM BACILLUS PS3: A REAPPRAISAL OF THE EFFECTS OF ATP AND MG2+

Abstract ATPase activity of the F 1 -ATPase from the thermophilic Bacillus PS3 (TF 1 ) was measured as a function of ATP concentration at three different magnesium ion concentrations. A high-performance chromatographic method was used to determine directly ADP concentration in the reaction medium and to measure the steady-state rate of its appearance. Multiphasic curves of ATPase activity versus ATP concentration were obtained, with a first saturating rate mode at low ATP concentrations, a higher rate mode which became predominant at ATP concentrations depending on magnesium concentration, and a marked inhibition of ATP hydrolysis at high ATP concentrations. These curves could be simulated with equivalent residual error either by assuming that the ATP-magnesium chelate is the substrate of the enzyme, free magnesium being an inhibitor, or that free ATP is the substrate, free magnesium being an essential activator. In both cases, the observed hydrolysis rate was assumed to be the sum of two independent rates with different kinetic parameters, as would be the case for an enzyme with functionally heterogeneous and independent catalytic sites. Cross-checking of the different series of kinetic parameters with the binding affinity of TF 1 for ATP, measured by a high-performance chromatographic method, is in favour of a model in which the hydrolysis rate is determined by the concentration of free ATP, free magnesium being an essential activator.

Investigating the structure of nucleotide binding sites on the chloroplast F1-ATPase using electron spin resonance spectroscopy

Abstract The relative structure and binding properties of nucleotide binding sites of the latent, nonactivated chloroplast F1(CF1)ATPase have been investigated by employing ESR spectroscopy using 2-N3-2′,3′-SL-ATP (2-N3-SL-ATP), a spin-labeled photoaffinity analog of ATP. These results are compared to data obtained in analogous experiments using CF1 that was either depleted of its e-subunit or activated by different methods. Nonactivated (na) CF1 in complex with 2-N3-SL-ATP exhibits ESR spectra typical for enzyme-bound spin labels. At increased 2-N3-SL-ATP concentration, a second spectral component for enzyme-bound spin label is observed. The line-shape of the second signal indicates an environment of the enzyme-bound radical that differs from the spin-labeled nucleotide bound first. It can be explained by an enzyme-bound radical bound in a way that allows for higher mobility, e.g. a nucleotide binding site in an “open” or “loose” conformation. Maximal binding of about 5 mol 2-N3-SL-ANP per mol of enzyme has been reached. Similar results are obtained when using enzyme that has been either previously depleted of the e-subunit or treated with the reducing agent dithiothreitol (DTT) in the cold. Upon heat-activation of CF1 in the presence of ATP and the presence or absence of the reducing agent DTT, the line-shape of the ESR spectra is observed to be quite different from the non-heat-treated enzyme forms. The “loose” or “open” nucleotide binding site described above (or at least an environment of the enzyme similar to this site) is observed to be accessible to 2-N3-SL-ATP even at substoichiometric concentrations of the nucleotide analog. The results presented indicate that the enzyme form of CF1 generated after heat treatment in the presence of ATP with or without DTT exhibits altered binding specificities mainly with respect to the sequence of occupation of two different types of nucleotide binding sites.

Cooperativity between the enzymatic sites of F1-ATPase revisited by the use of HPLC methods.

The fundamental question of the cooperativity between the enzymatic sites of F1-ATPase is examined in the light of new measurements of the enzymatic rate of ATP hydrolysis by CF1, the enzyme isolated from spinach chloroplasts. The experimental data, obtained with a chromatographic method, fit a model that involves two kinds of independent enzymatic sites working with metal-free ATP, with no need of cooperativity between the sites. Binding measurements between ADP or ATP and CF1 by the chromatographic method of Hummel and Dreyer (1962) also support this conclusion. The present data and interpretation are in agreement with those reported recently (Reynafarje and Pedersen, 1996) which show that the first order rate constant of ATP hydrolysis by MF1, the analogous enzyme from mitochondria, is virtually constant under experimental conditions involving either unisite or multisite hydrolysis of ATP. The present data and interpretation are discussed together with those reported previously, in particular with regard to the methods that were used to support the commonly accepted opposite viewpoint.

Nucleotide-CF1 interactions and current views on the catalytic mechanism

The binding of nucleotides on isolated subunits as well as on reconstituted CF1 core complex is reviewed. Nucleotide interaction with CF1 and consequent ATPase activity are always associated with the presence of Mg2+. The metal binding site studies using Electron Paramagnetic Resonance (EPR) and pulsed EPR conclude that the metal binding occurs prior to any nucleotide addition. The addition of nucleotide does not modify the enzymes metal binding site but brings on additional ligands with the phosphates of the nucleotides. The ATPase and nucleotide binding experiments with CF1 are also better interpreted by the hypothesis that Mg2+ is an activator rather than an inhibitor of the enzyme and that the actual substrate of CF1-ATPase is ATP rather than MgATP. The dual role of tentoxin as an inhibitor at low concentration (10-8-10-7 M) and activator at higher concentrations (10-6 M) of the enzymatic activity of CF1, is due to the presence of two different binding sites on CF1. The synthesis of a new cyclic analogue of tentoxin with alanine changed for a serine has shown that it was possible to dissociate the two roles. The serine tentoxin analogue has the same inhibition effect on CF1 but is no longer an activator. The binding of nucleotides may influence the stability, produce structural changes and, over long distance, cause movements of CF1. All these effects of nucleotide or metal binding and activation or inhibition of CF1 may help also to elucidate the role played by the catalytic and non catalytic sites. These questions are reviewed and analyzed with respect to the current views on the catalytic mechanism.

The Binding and Interaction of Pigments and Quinones in Bacterial Reaction Centers Studied by Infrared Spectroscopy

The primary photochemistry in bacterial photosynthesis involves a charge separation, stable for milliseconds to seconds, between specialized pigments acting as the primary electron donor and the intermediary electron acceptor, and quinones acting as primary and secondary electron acceptors. In bacterial reaction centers, the primary electron donor (P) is a bacteriochlorophyll (BChl) a or b dimer, the intermediary acceptor (H) is a bacteriopheophytin (BPheo) a or b monomer, and ubiquinones or menaquinones have been identified as electron acceptors (Q). The specifity, efficiency and stability of this charge separation relies on the arrangement and specific interaction of the pigments and redox components in the protein matrix. X-ray structures available for bacterial RC [1,2] provide a static picture of the quiescent state and suggest specific interactions of the pigments and quinones with their host site. However, additional information on the mechanisms and dynamics of primary electron transfer and on the concomitant change of interactions and protein conformation is required. Time-resolved optical spectroscopy (for a review, see [3]) as well as resonance Raman spectroscopy (for a review, see [4]) have provided such information.

Examination of catalytic activity of the β subunit isolated from chloroplast coupling factor 1

Abstract A simple and rapid procedure has been developed to isolate the subunit from chloroplast coupling factor 1 involving anion exchange HPLC. The β subunit was mainly in the monomeric form, along with some quantities of trimer and more complex aggregates of the protein. Decreasing pH down to neutral values or heating resulted in shifting the equilibrium in the medium to the formation of polymeric forms of the β subunit. By successive purification and fractionation of CF 1 and β subunit preparations, it was shown that the monomeric form of the β subunit catalysed ATP-ADP γ-phosphate exchange. However, it did not display ATPase or adenylate kinase activities. Aggregation of the β subunit was accompanied by irreversible inactivation of its ATP-ADP exchange activity.

Protein-prosthetic Group Interactions in Bacterial Reaction Centers

Specific interactions with their host polypeptides increasingly appear as essential in determining the excitation- and charge-transfer properties of the prosthetic groups within the bacterial reaction center. Detailed information on these interactions can be obtained from resonance Raman spectroscopy of the reaction center-bound pigments. Indeed, this technique currently permits selective observations of seven out of the eleven prosthetic groups born by wild-type reaction centers of Rhodospirillales. These Raman-observable groups are: i, the carotenoid (Lutz et al 1976, 1987), ii, the primary donor bacteriochlorophylls (Lutz & Robert 1985, Robert & Lutz 1986), iii, the accessory bacteriochlorophylls (Robert & Lutz 1987), iv, the normal acceptor bacteriopheophytin (Lutz 1980) and, v, the second bacteriopheophytin (Lutz 1980). Some recent, structural as well as functional results concerning these groups are outlined in the following.