Cation Binding to Halorhodopsin

A member of the retinal protein family, halorhodopsin, acts as an inward light-driven Cl^– pump. It was recently demonstrated that the <i>Natronomonas pharaonis</i> halorhodopsin-overproducing mutant strain KM-1 contains, in addition to the retinal chromophore, a lipid soluble chromophore, bacterioruberin, which binds to crevices between adjacent protein subunits. It is established that halorhodopsin has several chloride binding sites, with binding site I, located in the retinal protonated Schiff base vicinity, affecting retinal absorption. However, it remained unclear whether cations also bind to this protein. Our electron paramagnetic resonance spectroscopy examination of cation binding to the halorhodopsin mutant KM-1 reveals that divalent cations like Mn²⁺ and Ca²⁺ bind to the protein. Halorhodopsin has a high affinity for Mn²⁺ ions, which bind initially to several strong binding sites and then to binding sites that exhibit positive cooperativity. The binding behavior is pH-dependent, and its strength is influenced by the nature of counterions. Furthermore, the binding strength of Mn²⁺ ions decreases upon removal of the retinal chromophore from the protein or following bacterioruberin oxidation. Our results also indicate that Mn²⁺ ions, as well as Cl^– ions, first occupy binding sites other than site I. The observed synergetic effect between cation and anion binding suggests that while Cl^– anions bind to halorhodopsin at low concentrations, the occupancy of site I requires a high concentration

Electron Transport via Cytochrome C on Si–H Surfaces: Roles of Fe and Heme

Monolayers of the redox protein Cytochrome C (CytC) can be electrostatically formed on an H-terminated Si substrate, if the protein- and Si-surface are prepared so as to carry opposite charges. With such monolayers we study electron transport (ETp) via CytC, using a solid-state approach with macroscopic electrodes. We have revealed that currents via holo-CytC are almost 3 orders of magnitude higher than via the heme-depleted protein (→ apo-CytC). This large difference in currents is attributed to loss of the proteins’ secondary structure upon heme removal. While removal of only the Fe ion (→ porphyrin-CytC) does not significantly change the currents via this protein at room temperature, the 30–335 K temperature dependence suggests opening of a new ETp pathway, which dominates at high temperatures (>285 K). These results suggest that the cofactor plays a major role in determining the ETp pathway(s) within CytC

Doping Human Serum Albumin with Retinoate Markedly Enhances Electron Transport across the Protein

Electrons can migrate via proteins over distances that are considered long for nonconjugated systems. The nanoscale dimensions of proteins and their enormous structural and chemical flexibility makes them fascinating subjects for exploring their electron transport (ETp) capacity. One particularly attractive direction is that of tuning their ETp efficiency by “doping” them with small molecules. Here we report that binding of retinoate (RA) to human serum albumin (HSA) increases the solid-state electronic conductance of a monolayer of the protein by >2 orders of magnitude for RA/HSA ≥ 3. Temperature-dependent ETp measurements show the following with increasing RA/HSA: (a) The temperature-independent current magnitude of the low-temperature (<190 K) regime increases significantly (>300-fold), suggesting a decrease in the distance-decay constant of the process. (b) The activation energy of the thermally activated regime (>190 K) decreases from 220 meV (RA/HSA = 0) to 70 meV (RA/HSA ≥ 3)

Origin of Circular Dichroism of Xanthorhodopsin. A Study with Artificial Pigments

Xanthorhodopsin (xR) is a retinal protein that contains, in addition to the retinal moiety, a salinixanthin chromophore absorbing at 456, 486, and 520 nm [Balashov, S. P.; Science 2005, 309, 2061]. The CD spectrum of xR is very unique with a “conservative” character, containing negative and positive lobes and resembling the first derivative of the absorption spectrum [Balashov, S. P.; Biochemistry 2006, 45, 10998]. It was suggested that the CD spectrum is likely to be composed of several components and that the salinixanthin interacts closely with the retinal chromophore [Balashov, S. P.; Biochemistry 2006, 45, 10998; Imasheva, E. S.; Photochem. Photobiol. 2008, 84, 977; Lanyi, J. K.; Acta Bioenerg. 2008, 1777, 684; Smolensky, E.; Biochemistry 2009, 48, 8179; Smolensky Koganov, E.; Biochemistry 2013, 52, 1290]. In this work, we aim to further explore the nature and origin of the unique CD spectrum of xR. We follow the absorption and CD spectra at different pHs of wild-type (wt) xR and of artificial xR pigments, characterized by a shifted absorption maximum of the retinal chromophore, as well as their corresponding reduced retinal protonated Schiff base pigments. Our results revealed a protein residue (other than the protonated Schiff base counterion), for which protonation affects the CD spectrum by decreasing the negative lobe at ∼530 nm and the positive lobes at 478 and 455 nm, which might be due to elimination of excitonic coupling between the salinixanthin chromophores, although other possibilities cannot be completely excluded. This spectrum change occurs by the pH decreasing, even in artificial pigment where the absorption of the retinal pigment is significantly shifted from 570 to about 450 nm. The possible excitonic coupling between the salinixanthin chromophores and its contribution to the CD spectrum of xR were supported by a good fitting of the CD spectrum to conservative (excitonic) bands [Zsila, F.; Tetrahedron: Asymmetry 2001, 12, 3125; Zsila, F.; Tetrahedron: Asymmetry 2002, 13, 273]. We propose that the CD spectrum of xR consists of contributions from an excitonic coupling interaction between the salinixanthins chromophores located in different subunits of the 3D structure of xR, the chiral conformation of the salinixanthin within its binding site, and the contribution of the retinal chromophore to the negative lobe at around 550 nm

Retinal Binding to Apo-Gloeobacter Rhodopsin: The Role of pH and Retinal–Carotenoid Interaction

Over the past few decades, the structure, functions, properties, and molecular mechanisms of retinal proteins have been studied extensively. The newly studied retinal protein Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton from the cytoplasmic region to the extracellular region of a cell following light absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In the present study, we report the effect of pH on the binding of retinal to the apo-protein of gR, in the presence and absence of sal, to form the gR pigment. We found that binding at different pH levels reflects the titration of two different protein residues, one at the lower p<i>K</i>_a 3.5 and another at the higher p<i>K</i>_a 8.4, that affect the pigment’s formation. The maximum amount of pigment was formed at pH 5, both with and without the presence of sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190. Furthermore, it is suggested that occupation of the binding site by the retinal chromophore induces protein conformational alterations which in turn affect the carotenoid conformation, which precedes the formation of the retinal–protein covalent bond. Our examination of synthetic retinal analogues in which the ring structure was modified revealed that, in the absence of sal, the retinal ring structure affects the rate of pigment formation and that the intact structure is needed for efficient pigment formation. However, the presence of sal abolishes this effect, and all-trans retinal and its modified ring analogues bind at a similar rate

Light-Controlled Spin Filtering in Bacteriorhodopsin

The role of the electron spin in chemistry and biology has received much attention recently owing to to the possible electromagnetic field effects on living organisms and the prospect of using molecules in the emerging field of spintronics. Recently the chiral-induced spin selectivity effect was observed by electron transmission through organic molecules. In the present study, we demonstrated the ability to control the spin filtering of electrons by light transmitted through purple membranes containing bacteriorhodopsin (bR) and its D96N mutant. The spin-dependent electrochemical cyclic voltammetry (CV) and chronoamperometric measurements were performed with the membranes deposited on nickel substrates. High spin-dependent electron transmission through the membranes was observed; however, after the samples were illuminated by 532 nm light, the spin filtering in the D96N mutant was dramatically reduced whereas the light did not have any effect on the wild-type bR. Beyond demonstrating spin-dependent electron transmission, this work also provides an interesting insight into the relationship between the structure of proteins and spin filtering by conducting electrons

Temperature-Dependent Solid-State Electron Transport through Bacteriorhodopsin: Experimental Evidence for Multiple Transport Paths through Proteins

Electron transport (ETp) across bacteriorhodopsin (bR), a natural proton pump protein, in the solid state (dry) monolayer configuration, was studied as a function of temperature. Transport changes from thermally activated at <i>T</i> > 200 K to temperature independent at <130 K, similar to what we have observed earlier for BSA and apo-azurin. The relatively large activation energy and high temperature stability leads to conditions where bR transports remarkably high current densities above room temperature. Severing the chemical bond between the protein and the retinal polyene only slightly affected the main electron transport via bR. Another thermally activated transport path opens upon retinal oxime production, instead of or in addition to the natural retinal. Transport through either or both of these paths occurs on a background of a general temperature-independent transport. These results lead us to propose a generalized mechanism for ETp across proteins, in which tunneling and hopping coexist and dominate in different temperature regimes

Conjugated Cofactor Enables Efficient Temperature-Independent Electronic Transport Across ∼6 nm Long Halorhodopsin

We observe temperature-independent electron transport, characteristic of tunneling across a ∼6 nm thick Halorhodopsin (phR) monolayer. phR contains both retinal and a carotenoid, bacterioruberin, as cofactors, in a trimeric protein-chromophore complex. This finding is unusual because for conjugated oligo-imine molecular wires a transition from temperature-independent to -dependent electron transport, ETp, was reported at ∼4 nm wire length. In the ∼6 nm long phR, the ∼4 nm 50-carbon conjugated bacterioruberin is bound parallel to the α-helices of the peptide backbone. This places bacterioruberin’s ends proximal to the two electrodes that contact the protein; thus, coupling to these electrodes may facilitate the activation-less current across the contacts. Oxidation of bacterioruberin eliminates its conjugation, causing the ETp to become temperature dependent (>180 K). Remarkably, even elimination of the retinal-protein covalent bond, with the fully conjugated bacterioruberin still present, leads to temperature-dependent ETp (>180 K). These results suggest that ETp via phR is cooperatively affected by both retinal and bacterioruberin cofactors

Probing Ultrafast Photochemistry of Retinal Proteins in the Near-IR: Bacteriorhodopsin and Anabaena Sensory Rhodopsin vs Retinal Protonated Schiff Base in Solution

Photochemistry of bacteriorhodopsin (bR), anabaena sensory rhodopsin (ASR), and all-trans retinal protonated Schiff base (RPSB) in ethanol is followed with femtosecond pump–hyperspectral near-IR (NIR) probe spectroscopy. This is the first systematic probing of retinal protein photochemistry in this spectral range. Stimulated emission of the proteins is demonstrated to extend deep into the NIR, and to decay on the same characteristic time scales previously determined by visible probing. No signs of a transient NIR absorption band above λ_pr > 1.3 μm, which was recently reported and is verified here for the RPSB in solution, is observed in either protein. This discrepancy demonstrates that the protein surroundings change photochemical traits of the chromophore significantly, inducing changes either in the energies or couplings of photochemically relevant electronic excited states. In addition, low-frequency and heavily damped spectral modulations are observed in the NIR signals of all three systems up to 1.4 μm. By background subtraction and Fourier analysis they are shown to resemble wave packet signatures in the visible, stemming from multiple vibrational modes and by analogy are assigned to torsional wave packets in the excited state of the retinal chromophore. Differences in the vibrational frequencies between the three samples and the said discrepancy in transient spectra are discussed in terms of opsin effects on the RPSB electronic structure

Bacteriorhodopsin/Ag Nanoparticle-Based Hybrid Nano-Bio Electrocatalyst for Efficient and Robust H₂ Evolution from Water

Searching for novel hybrid electrocatalysts with high activity and strong durability for a direct electrochemical hydrogen evolution reaction (HER) is extremely desirable but still remains a significant challenge. Herein, we report a novel solid carbon cloth-supported hybrid nano-bio electrocatalyst, decorated with Ag nanoparticles and proton-pumping bacteriorhodopsin (bR) (Ag/bR/CP) that were prepared by in situ electroless deposition and vesicle fusion technology, respectively. When applied as a hydrogen evolution cathode, the Ag/bR/CP shows a low onset overpotential of 63 mV, good durability (no detectable change in its catalytic activity for up to 1000 cycles in alkaline media), and enhanced HER performance under 550 nm irradiation, attributed to the activation of Ag and synergistic effects following light absorption, demonstrated by photoelectrochemical measurements