László Zimányi

THE TWO CONSECUTIVE M SUBSTATES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN ARE AFFECTED SPECIFICALLY BY THE D85N AND D96N RESIDUE REPLACEMENTS

Abstract— The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Váró and Lanyi, Biochemistry30, 5016–5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild‐type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M, but not M2 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre‐switch and post‐switch states, respectively.

Anion binding to the chloride pump, halorhodopsin, and its implications for the transport mechanism

The light‐driven chloride pump, halorhodopsin, binds and transports chloride across the membrane, and to a lesser extent nitrate. Binding and transport kinetics, and resonance Raman spectra of the retinal Schiff base, with these anions suggest the existence of two mutually exclusive binding sites. One of these may be the uptake site, and the other the release site during the transport. Plausible locations can be suggested for these sites, because halorhodopsin is a small protein with few buried positively charged residues, and the primary structure of a second pigment with similar function has recently become available for comparison.

Configuration of the light induced electric field in thylakoid and its possible role in the kinetics of the 515 nm absorbance chance

Abstract Theoretical calculations of the electric potential were carried out using a model of the thylakoid consisting of a spherical dielectric membrane surrounded both inside and outside by highly conductive material. The calculations yielded typical configurations and intensities of the electrical field induced by charges either localized in the membrane or delocalized in the conductive phases. It is shown that the build-up of the uniform transmembrane field is strictly correlated with translocation of charges from the membrane onto the boundaries of the conductive phases which induces a considerable increment in the field-intensity over the greater part of the thylakoid. This shows that the slow rise of the electrochromic absorbance change may be physically related to the slow translocation of charges from the membrane into the conductive phases which is linked to rate-limiting electron transport processes.

Porous Silicon/Photosynthetic Reaction Center Hybrid Nanostructure

The purified photosynthetic reaction center protein (RC) from Rhodobacter sphaeroides R-26 purple bacteria was bound to porous silicon microcavities (PSiMc) either through silane-glutaraldehyde (GTA) chemistry or via a noncovalent peptide cross-linker. The characteristic resonance mode in the microcavity reflectivity spectrum red shifted by several nanometers upon RC binding, indicating the protein infiltration into the porous silicon (PSi) photonic structure. Flash photolysis experiments confirmed the photochemical activity of RC after its binding to the solid substrate. The kinetic components of the intraprotein charge recombination were considerably faster (τ(fast) = 14 (±9) ms, τ(slow) = 230 (±28) ms with the RC bound through the GTA cross-linker and only τ(fast) = 27 (±3) ms through peptide coating) than in solution (τ(fast) = 120 (±3) ms, τ(slow) = 1387 (±2) ms), indicating the effect of the PSi surface on the light-induced electron transfer in the protein. The PSi/RC complex was found to oxidize the externally added electron donor, mammalian cytochrome c, and the cytochrome oxidation was blocked by the competitive RC inhibitor, terbutryne. This fact indicates that the specific surface binding sites on the PSi-bound RC are still accessible to external cofactors and an electronic interaction with redox components in the aqueous environment is possible. This new type of biophotonic material is considered to be an excellent model for new generation applications at the interface of silicon-based electronics and biological redox systems designed by nature.

A residue substitution near the beta-ionone ring of the retinal affects the M substates of bacteriorhodopsin

The switch in the bacteriorhodopsin photocycle, which reorients access of the retinal Schiff base from the extracellular to the cytoplasmic side, was suggested to be an M1----M2 reaction (Váró and Lanyi. 1991. Biochemistry. 30:5008-5015, 5016-5022). Thus, in this light-driven proton pump it is the interconversion of proposed M substates that gives direction to the transport. We find that in monomeric, although not purple membrane-lattice immobilized, D115N bacteriorhodopsin, the absorption maximum of M changes during the photocycle: in the time domain between its rise and decay it shifts 15 nm to the blue relative to the spectrum at earlier times. This large shift strongly supports the existence of two M substates. Since D115 is located near the beta-ionone ring of the retinal, the result raises questions about the possible involvement of the retinal chain or protein residues as far away as 10 A from the Schiff base in the mechanism of the switching reaction.

Effect of CO2 on the energization of thylakoids in leaves of higher plants

We investigated the effect of CO2 on the flash‐induced electrochromic absorbance change of chloroplasts in leaves of higher plants. In leaves depleted of CO2 the initial electrochromic rise was followed by a fast (t 10–20 ms) and a slow (100–200 ms) decay. These kinetic components could be correlated with a dissipative and an ATP‐synthetizing current, respectively. In leaves supplied with CO2 an additional slow electrochromic rise (5–10 ms) appeared in the absorbance change. This component could be tentatively correlated with the enhancement of the electron transport by CO2 between the two photosystems. In leaves supplied with CO2 the decay could be fitted with a single exponential with a t ≈200 ms. We conclude that both energization and ATP‐synthesis are strongly regulated by CO2 in chloroplasts in situ.

Disentangling Electron Tunneling and Protein Dynamics of Cytochrome c through a Rationally Designed Surface Mutation

Nonexponential distance dependence of the apparent electron-transfer (ET) rate has been reported for a variety of redox proteins immobilized on biocompatible electrodes, thus posing a physicochemical challenge of possible physiological relevance. We have recently proposed that this behavior may arise not only from the structural and dynamical complexity of the redox proteins but also from their interplay with strong electric fields present in the experimental setups and in vivo (J. Am Chem. Soc. 2010, 132, 5769-5778). Therefore, protein dynamics are finely controlled by the energetics of both specific contacts and the interaction between the proteins dipole moment and the interfacial electric fields. In turn, protein dynamics may govern electron-transfer kinetics through reorientation from low to high donor-acceptor electronic coupling orientations. Here we present a combined computational and experimental study of WT cytochrome c and the surface mutant K87C adsorbed on electrodes coated with self-assembled monolayers (SAMs) of varying thickness (i.e., variable strength of the interfacial electric field). Replacement of the positively charged K87 by a neutral amino acid allowed us to disentangle protein dynamics and electron tunneling from the reaction kinetics and to rationalize the anomalous distance dependence in terms of (at least) two populations of distinct average electronic couplings. Thus, it was possible to recover the exponential distance dependence expected from ET theory. These results pave the way for gaining further insight into the parameters that control protein electron transfer.

Fast Redox Perturbation of Aqueous Solution by Photoexcitation of Pyranine

Abstract— Intense illumination (60‐120 MW/cm2) of an oxygen‐free aqueous solution of pyranine (8‐hydroxypyrene‐l,3,6‐tri‐sulfonate) by the third harmonic frequency of an Nd‐Yag laser (355 nm) drives a two successive‐photon oxidative process of the dye. The first photon excites the dye to its first electronic singlet state. The second photon interacts with the excited molecule, ejects an electron to the solution and deactivates the molecule to a ground state of the oxidized dye (φ+). The oxidized product, φ+, is an intensely colored compound (Λmax= 445 nm, ε= 43 000 ± 1000 M−1 cm−1) that reacts with a variety of electron donors like quinols, ascorbate and ferrous compounds. In the absence of added reductant, φ+ is stable, having a lifetime of ‐10 min. In acidic solutions the solvated electrons generated by the photochemical reaction react preferentially with H+. In alkaline solution the favored electron acceptor is the ground‐state pyranine anion and a radical, φ, of the reduced dye is formed. The reduced product is well distinguished from the oxidized one, having its maximal absorption at 510 nm with e = 25 000 ± 2000 M‐l cm−1. The oxidized radical can be reduced either by φ‐ or by other electron donors. The apparent second‐order rate constants of these reactions, which vary from 106 up to 109M−1 s−1, are slower than the rates of diffusion‐controlled reactions. Thus the redox reactions are limited by an energy barrier for electron transfer within the encounter complex between the reactants.

Assembly of Purple Membranes on Polyelectrolyte Films

The membrane protein bacteriorhodopsin in its native membrane bound form (purple membrane) was adsorbed and incorporated into polyelectrolyte multilayered films, and adsorption was in situ monitored by optical waveguide light-mode spectroscopy. The formation of a single layer or a double layer of purple membranes was observed when adsorbed on negatively or positively charged surfaces, respectively. The purple membrane patches adsorbed on the polyelectrolyte multilayers were also evidenced by atomic force microscopy images. The driving forces of the adsorption process were evaluated by varying the ionic strength of the solution as well as the purple membrane concentration. At high purple membrane concentration, interpenetrating polyelectrolyte loops might provide new binding sites for the adsorption of a second layer of purple membranes, whereas at lower concentrations only a single layer is formed. Negative surfaces do not promote a second protein layer adsorption. Driving forces other than just electrostatic ones, such as hydrophobic forces, should play a role in the polyelectrolyte/purple membrane layering. The subtle interplay of all these factors determines the formation of the polyelectrolyte/purple membrane matrix with a presumably high degree of orientation for the incorporated purple membranes, with their cytoplasmic, or extracellular side toward the bulk on negatively or positively charged polyelectrolyte, respectively. The structural stability of bacteriorhodopsin during adsorption onto the surface and incorporation into the polyelectrolyte multilayers was investigated by Fourier transform infrared spectroscopy in attenuated total reflection mode. Adsorption and incorporation of purple membranes within polyelectrolyte multilayers does not disturb the conformational majority of membrane-embedded alpha-helix structures of the protein, but may slightly alter the structure of the extramembraneous segments or their interaction with the environment. This high stability is different from the lower stability of the predominantly beta-sheet structures of numerous globular proteins when adsorbed onto surfaces.

Configuration of the electric field and distribution of ions in energy transducing biological membranes: model calculations in a vesicle containing discrete charges

Configuration of the electric field and distribution of ions were calculated in a vesicle adopted to model energy transducing biological membranes. The model consists of a spherical, homogeneous low dielectric membrane and electrolyte inside and outside the sphere. The electric potential induced by discrete charges, monopoles and dipoles embedded in the membrane, was calculated for vesicles with different dielectric constants and ion concentrations of the electrolyte. We show that charges embedded in the membrane induce an electric and ionic microcompartmentalization in the vesicle: they generate a strong local field in their vicinity both in the membrane and the aqueous phases and also create a uniform transmembrane “delocalized” field in the rest of the vesicle. These calculations yield quantitative estimates for local electric effects assumed to be important in the operation of electron and proton transport and in the coupling between the electron transport system and the ATP-synthetase in oxidative and photosynthetic energy transduction.