Retinal-Salinixanthin Interactions in a Thermophilic Rhodopsin.

In microbial rhodopsins (also called retinal proteins), the retinal chromophore is used for harvesting light. A carotenoid molecule has been reported to complement the retinal as light harvesting antenna in bacterial retinal proteins, although examples are scarce. In this paper, we present the formation of a novel antenna complex between thermophilic rhodopsin (TR) and the carotenoid salinixanthin (Sal). The complex formation and its structure were studied using UV-visible absorption as well as circular dichroism (CD) spectroscopies. Our studies indicate that the complex is formed in both the trimeric and monomeric forms of TR. CD spectroscopy suggests that excitonic coupling takes place between retinal and Sal. The binding of Sal with artificial TR pigments derived from synthetic retinal analogues further supports the contribution of the retinal chromophore to the CD spectrum. These studies further support the possibility of interaction between the 4-keto ring of the Sal and the retinal in TR-Sal complexes. Temperature-dependent CD spectra indicate that the positive band (ca. 482 nm) of the bisignate CD spectra of the studied complexes originates from the contribution of excitonic coupling and induced chirality of Sal in the protein binding site. The presence of a relatively smaller glycine residue in the vicinity of the retinal chromophore in TR is proposed to be crucial for binding with Sal. The results are expected to shed light on the mechanism of retinal-carotenoid interactions in other biological systems.

Direct evidence for heme-assisted solid-state electronic conduction in multi-heme c-type cytochromes.

Multi-heme cytochrome c (Cytc) proteins are key for transferring electrons out of cells, to enable intracellular oxidation to proceed in the absence of O2. In these proteins most of the hemes are arranged in a linear array suggesting a facile path for electronic conduction. To test this, we studied solvent-free electron transport across two multi-heme Cytc-type proteins: MtrF (deca-heme Cytc) and STC (tetra-heme Cytc). Transport is measured across monolayers of these proteins in a solid state configuration between Au electrodes. Both proteins showed 1000× higher conductance than single heme, or heme-free proteins, but similar conductance to monolayers of conjugated organics. Conductance is found to be temperature-independent (320-80 K), suggesting tunneling as the transport mechanism. This mechanism is consistent with I-V curves modelling, results of which could be interpreted by having protein-electrode coupling as rate limiting, rather than transport within the proteins.

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 pKa 3.5 and another at the higher pKa 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.

Efficient femtosecond energy transfer from carotenoid to retinal in gloeobacter rhodopsin-salinixanthin complex.

The retinal proton pump xanthorhodopsin (XR) was recently found to function with an attached carotenoid light harvesting antenna, salinixanthin (SX). It is intriguing to discover if this departure from single chromophore architecture is singular or if it has been adopted by other microbial rhodopsins. In search of other cases, retinal protein encoding genes in numerous bacteria have been identified containing sequences corresponding to carotenoid binding sites like that in XR. Gloeobacter rhodopsin (GR), exhibiting particularly close homology to XR, has been shown to attach SX, and fluorescence measurements suggest SX can function as a light harvesting (LH) antenna in GR as well. In this study, we test this suggestion in real time using ultrafast transient absorption. Results show that energy transfer indeed occurs from S2 of SX to retinal in the GR-SX composite with an efficiency of ∼40%, even higher than that in XR. This validates the earlier fluorescence study, and supports the notion that many microbial retinal proteins use carotenoid antennae to harvest light.

Asymmetric toggling of a natural photoswitch: ultrafast spectroscopy of Anabaena sensory rhodopsin.

Photochemistry in retinal proteins (RPs) is determined both by the properties of the retinal chromophore and by its interactions with the surrounding protein. The initial retinal configuration, and the isomerization coordinates active in any specific protein, must be important factors influencing the course of photochemistry. This is illustrated by the vast differences between the photoisomerization dynamics in visual pigments which start 11-cis and end all-trans, and those observed in microbial ion pumps and sensory rhodopsins which start all-trans and end in a 13-cis configuration. However, isolating these factors is difficult since most RPs accommodate only one active stable ground-state configuration. Anabaena sensory rhodopsin, allegedly functioning in cyanobacteria as a wavelength sensor, exists in two stable photoswitchable forms, containing all-trans and 13-cis retinal isomers, at a wavelength-dependent ratio. Using femtosecond spectroscopy, and aided by extraction of coherent vibrational signatures, we show that cis-to-trans photoisomerization, as in visual pigments, is ballistic and over in a fraction of a picosecond, while the reverse is nearly 10 times slower and kinetically reminiscent of other microbial rhodopsins. This provides a new test case for appreciating medium effects on primary events in RPs.

Deciphering excited state evolution in halorhodopsin with stimulated emission pumping.

The primary photochemical dynamics of Hb. pharaonis Halorhodopsin (pHR) are investigated by femtosecond visible pump-near IR dump-hyperspectral probe spectroscopy. The efficiency of excited state depletion is deduced from transient changes in absorption, recorded with and without stimulated emission pumping (SEP), as a function of the dump delay. The concomitant reduction of photocycle population is assessed by probing the "K" intermediate difference spectrum. Results show that the cross section for stimulating emission is nearly constant throughout the fluorescent state lifetime. Probing "K" demonstrates that dumping produces a proportionate reduction in photocycle yields. We conclude that, despite its nonexponential internal conversion (IC) kinetics, the fluorescent state in pHR constitutes a single intermediate in the photocycle. This contrasts with conclusions drawn from the study of primary events in the related chloride pump from Hb. salinarum (sHR), believed to produce the "K" intermediate from a distinct short-lived subpopulation in the excited state. Our discoveries concerning internal conversion dynamics in pHR are discussed in light of recent expectations for similar excited state dynamics in both proteins.