M. Leonhard

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.

Electrochemical redox titration of cofactors in the reaction center from Rhodobacter sphaeroides

The electrochemical redox poising of the primary electron donor P and of the quinone electron acceptor(s) Q in isolated reaction centers from Rhodobacter sphaeroides in an ultra‐thin‐layer electrochemical cell, monitored by chronoamperometry and by spectroscopy in the visible/near‐infrared region, is reported. Electrical application of a redox potential of +0.4 V (vs. Ag/AgCl/3 M KCl) leads to quantitative formation of the π‐cation radical of P within a few minutes. The oxidized product can be re‐reduced to the neutral species by application of 0 V, and full reversibility is maintained over many‐cycles. By poising at a series of intermediate potentials, a titration curve for the 865 nm P band was obtained, which could be fitted to a Nernst function with E m = 0.485 vs. SHE and n = 0.96. By Application of negative potentials (−0.2 V and −0.45 V vs. Ag/AgCl/3 M KCl), the quinone electron acceptors were reversibly reduced as demonstrated by the shift of bacteriopheophytin absorption and drastically changed kinetics of charge recombination. The use of this thin‐layer electrochemical technique for the determination of midpoint potentials, for the investigation of redox‐poised electron transfer reactions as well as for spectroscopy in the mid‐infrared region is discussed.

A physical characterization of some detergents of potential use for membrane protein crystallization

Micellar solutions of lauryldimethylamine oxide, n‐dodecyl‐β‐D‐maltoside and 1‐dodecanoylpropanediol‐3‐phosphorylcholine were studied by use of small‐angle neutron scattering. These detergents have been selected due to their use as solubilizing agents for membrane proteins. LDAO was found to form a homogeneous, approximately spherical micelle with a radius of 20.7 Å and an M r of 16 000. N‐Dodecyl‐β‐D‐maltoside forms an inhomogeneous micelle with a core of low scattering density surrounded by a shell of high scattering density. The data are in accord with a micelle forming an oblate ellipsoid and the disaccharide group pointing outward radially from the hydrophobic group. The semi‐axes are 16.8 and 25.5 Å and the M r is 66 000. 1‐Dodecanoylpropanediol‐3‐phosphorylcholine forms a rather homogeneous, roughly spherical micelle. The radius is 24 Å, the M r being 28 700. The data indicate a tangential packing of the phosphorylcholine head groups into a polar layer of 3–4 Å surrounding the micelle core. The use of these detergents as solubilizing agents during membrane protein crystallization is discussed.

Binding and interaction of the primary and the secondary electron acceptor quinones in bacterial photosynthesis: An infrared spectroelectrochemical study of Rhodobacter sphaeroides reaction centers

The vibrational modes of the primary and secondary electron acceptors QA and QB, their semiquinone anions and their respective protein environment in Rhodobacter sphaeroides reaction centers have been characterized using combinations of electrochemically-induced and light-induced Fourier-transform infrared (IR) difference spectroscopy. Q−A/QA and Q−B/QB IR difference spectra without contributions of other cofactors were generated by three different methods: (1) electrochemically, by reduction of QA to Q−A; (2) photochemically, with flash-induced formation of P+Q−A or P+Q−B, rereduction of P+ by cytochrome (cyt) c2, and electrochemical rereduction of cyt c2; (3) photochemically and electrochemically, by subtraction of redox-induced P+/P difference spectra from light-induced P+Q−A/PQA and P+Q−B/PQB difference spectra. Although Q−A was generated by completely different methods, and in one case (3) a charge-separated state is involved, almost identical Q−A/QA and only slightly different Q−B/QB difference spectra have been obtained. Bands at 1630 cm−1 and 1640 cm−1 are proposed as candidates for the C=O modes of QA and QB, respectively. The C-O modes of Q−A and Q−B are assigned to bands at 1462 cm−1 and 1478-88 cm−1, respectively. Difference bands at 1668 cm−1 and 1652 cm−1 in Q−A/QA difference spectra are more likely to arise from amide-I modes or side chain vibrations of amino acids to which QA is hydrogen-bonded. A number of difference bands between 1520 cm−1 and 1560 cm−1 possibly arise from amide-II vibrations and aromatic amino-acid side chain residues in the vicinity of QA and QB. A differential feature at 1734 cm−11726 cm−1 in Q−A/QA difference spectra probably arises from changes in the protonation state or environment of distant carboxyl groups. An alternative explanation in terms of changes in the environment of the 10a ester C=O group of bacteriopheophytin L, however, cannot be excluded. Bands between 1724 cm−1 and 1740 cm−1 in Q−B/QB difference spectra are tentatively assigned to a protonation of ASP L213 and/or a change in the environment of GLU L212, both being located in the vicinity of QB and involved in the proton transfer to QB (Okamura, M.Y. and Feher, G. (1992) Annu. Rev. Biochem. 61, 861–896).

Stabilization of detergent-solubilized Ca2+-ATPase by poly(ethylene glycol)

The (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum (SR) has been solubilized with 1-alkanoyl propanediol-3-phosphorylcholines with chainlengths ranging between 8 and 12 C atoms. A marked dependence of the ATPase activity upon the chainlength was found, indicating that alkyl chainlengths with 12 C atoms are necessary for retention of activity. Addition of poly(ethylene glycol) to the eluting buffers used for gel filtration of the ATPase-detergent micelles was found to increase the activity and the long-term stability significantly. In the presence of Ca2+, the elution volume indicated an ATPase dimer, whereas in the absence of Ca2+ the elution volume indicated a monomeric solution. The purity of the preparations after gel filtration was improved by subsequent chromatography with a hydroxyapatite column.

Model Compound Studies of Pigments Involved in Photosynthetic Energy Conversion: Infrared (IR)-Spectro-Electrochemistry of Chlorophylls and Pheophytins

In the primary reactions of photosynthesis, the light-induced charge separation and stabilization involves the generation of a radical cation and of radical anions at the various steps of electron transfer. In the reaction centers, specialized pigment molecules and redox components act as primary electron donors and electron acceptors. They are embedded in the protein matrix and provided with very specific interactions that seem to be responsible for the spectral and redox properties of the pigments as well as for the efficiency and specificity of electron transfer.

Infrared Difference Spectroscopy of Electro-chemically Generated Redox States in Bacterial Reaction Centers

In bacterial photosynthetic reaction centers (RC), about 40% of the energy of the absorbed photon or trapped exciton are stored in a charge-separated state, whose free energy can be used by secondary redox reactions. This charge-separated state is created in a sequence of steps; i.e. an electron is transferred from the primary electron donor P (a bacteriochlorophyll dimer, BChl2) via a monomeric bacteriochlorophyll B to an intermediary electron acceptor H (a bacteriopheophytin, BPheo), and from there to a primary and secondary quinone acceptor QA and QB, thus creating a P+Q− state (for a review, see [1]). The free energy stored in this state can be expressed as the sum of the energy needed to shift the redox poise of P/P+ and of Q/Q− from the quiescent state of the RC, where both P and Q are neutral, to an accumulation of the charged radicalic species.

Infrared Difference Spectroscopy of Pigments and Redox Components in the Bacterial Reaction Center: Comparison with Model Compounds

The primary photochemistry in bacterial photosynthesis involves a charge separation, stable for milliseconds to seconds, between specialized pigments and redox components. In bacterial reaction centers (RC), the identity and spatial arrangement of these components within the protein matrix is known from crystallographic and spectroscopic data. The specifity, efficiency and stability of primary charge separation relies on specific interactions of the pigments and redox components with the protein. These interactions are able to support different redox states of the pigments and quinones via different binding properties. While the X-ray structures available for bacterial RC provide a static picture of the quiescent state and suggest specific interactions of the pigments and quinones with their host site, additional information on the mechanisms and the dynamics of primary electron transfer, with the concomitant change of interactions and protein conformation, is needed from spectroscopic techniques.

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.

Characterization of P700 by FTIR Difference Spectroscopy

The primary photochemical act in photosynthesis leads to the generation of the radical cation of a specialized chlorophyll (Chl) or bacteriochlorophyll (BChl) species. For purple bacteria, the primary electron donor is a pair of BChl whose molecular structure has been extensively investigated by various spectroscopic techniques and recently elucidated by X-ray cristallography of the bacterial reaction center. In the absence of high-resolution X-ray data for green plant PS I and PS II, proposals for the structure and bonding interactions of their primary donor rely only on spectroscopy. In particular, the primary donor of PS I, P700, is probably a Chl a dimer, although several spectroscopic studies suggest that the positive charge in the P700+ radical cation is localized on only one of the two Chl molecules that comprise P700 (1–3). Model studies also incorporate a possible keto-enol tautomerization of Chl a upon P700 photooxidation (4,5).