Protonation reactions and their coupling in bacteriorhodopsin

Light-induced changes of the proton affinities of amino acid side groups are the driving force for proton translocation in bacteriorhodopsin. Recent progress in obtaining structures of bacteriorhodopsin and its intermediates with an increasingly higher resolution, together with functional studies utilizing mutant pigments and spectroscopic methods, have provided important information on the molecular architecture of the proton transfer pathways and the key groups involved in proton transport. In the present paper I consider mechanisms of light-induced proton release and uptake and intramolecular proton transport and mechanisms of modulation of proton affinities of key groups in the framework of these data. Special attention is given to some important aspects that have surfaced recently. These are the coupling of protonation states of groups involved in proton transport, the complex titration of the counterion to the Schiff base and its origin, the role of the transient protonation of buried groups in catalysis of the chromophore's thermal isomerization, and the relationship between proton affinities of the groups and the pH dependencies of the rate constants of the photocycle and proton transfer reactions.     

pH dependence of light-driven proton pumping by an archaerhodopsin from Tibet: comparison with bacteriorhodopsin

The pH-dependence of photocycle of archaerhodopsin 4 (AR4) was examined, and the underlying proton pumping mechanism investigated. AR4 is a retinal-containing membrane protein isolated from a strain of halobacteria from a Tibetan salt lake. It acts as a light-driven proton pump like bacteriorhodopsin (BR). However, AR4 exhibits an "abnormal" feature--the time sequence of proton release and uptake is reversed at neutral pH. We show here that the temporal sequence of AR4 reversed to "normal"--proton release preceding proton uptake--when the pH is increased above 8.6. We estimated the pK(a) of the proton release complex (PRC) in the M-intermediate to be approximately 8.4, much higher than 5.7 of wide-type BR. The pH-dependence of the rate constant of M-formation shows that the pK(a) of PRC in the initial state of AR4 is approximately 10.4, whereas it is 9.7 in BR. Thus in AR4, the chromophore photoisomerization and subsequent proton transport from the Schiff base to Asp-85 is coupled to a decrease in the pK(a) of PRC from 10.4 to 8.4, which is 2 pK units less than in BR (4 units). This weakened coupling accounts for the lack of early proton release at neutral pH and the reversed time sequence of proton release and uptake in AR4. Nevertheless the PRC in AR4 effectively facilitates deprotonation of primary proton acceptor and recovery of initial state at neutral pH. We found also that all pK(a)s of the key amino acid residues in AR4 were elevated compared to those of BR.     

Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration

The recent discovery of a carotenoid light-harvesting antenna in xanthorhodopsin, a retinal-based proton pump in Salinibacter ruber, made use of photoinhibition of respiration in whole cells to obtain action spectra [Balashov et al. Science 309, (2005) 2061-2064]. Here we provide further details of this phenomenon, and compare action spectra in three different systems where carotenoids have different functions or efficiencies of light-harvesting. The kinetics of light-induced inhibition of respiration in Salinibacter ruber was determined with single short flashes, and the photochemical cross section of the photoreaction was estimated. These measurements confirm that the xanthorhodopsin complex includes no more than a few, and most likely only one, carotenoid molecule, which is far less than the core complex antenna of photosynthetic bacteria. Although the total cross-section of light absorption in the purple bacterium Rhodospirillum rubrum greatly exceeds that in Salinibacter, the cross-sections are roughly equivalent in the shared wavelength range. We show further that despite interaction of bacterioruberin with archaerhodopsin, another retinal-based proton pump, there is no significant energy transfer from this carotenoid. This emphasizes the uniqueness of the salinixanthin-retinal interaction in xanthorhodopsin, and indicates that bacterioruberin in Halorubrum species has a structural or photoprotective rather than energetic role.     

Induced chirality of the light-harvesting carotenoid salinixanthin and its interaction with the retinal of xanthorhodopsin

In xanthorhodopsin, a retinal protein-carotenoid complex of Salinibacter ruber, the carotenoid salinixanthin functions as a light-harvesting antenna in supplying additional excitation energy for retinal isomerization and proton transport. Another retinal protein, archaerhodopsin, has been shown to contain a carotenoid, bacterioruberin, but without an antenna function. We report here that the binding site confers a chiral geometry on salinixanthin in xanthorhodopsin and confirm that the same is true for bacterioruberin in archaerhodopsin. Cell membranes containing these rhodopsins exhibit CD spectra with sharp positive bands in the visible region where the carotenoids absorb, and in the case of xanthorhodopsin a negative band at 536 nm, as well as bands in the UV region. The carotenoid in ethanol has very weak optical activity in the visible region of the spectrum. Denaturation of the opsin upon deprotonation of the Schiff base at pH 12.5 eliminates the induced CD bands in both proteins. In one of these proteins, but not in the other, the carotenoid binding site depends entirely on the retinal. Hydrolysis of the retinal Schiff base of xanthorhodopsin with hydroxylamine eliminates the induced CD bands of salinixanthin. In contrast, hydrolysis of the Schiff base in archaerhodopsin does not abolish the CD bands of bacterioruberin. Thus, consistent with its antenna function, the carotenoid binding site interacts closely with the retinal only in xanthorhodopsin, and this interaction is the major source of the CD bands. In this protein, protonation of the counterion with a decrease in pH from 8 to 5 causes significant changes in the CD spectrum. The observed spectral features suggest that binding of salinixanthin in xanthorhodopsin involves the cyclohexenone ring of the carotenoid and its conformational heterogeneity is restricted.     

Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells.

Translational stress-induced mutagenesis (TSM) refers to the mutator phenotype observed in Escherichia coli cells expressing a mutant allele (mutA or mutC) of the glycine tRNA gene glyV (or glyW). Because of an anticodon mutation, expression of the mutA allele results in low levels of Asp-->Gly mistranslation. The mutA phenotype does not require lexA-regulated SOS mutagenesis functions, and appears to be suppressed in cells defective for RecABC-dependent homologous recombination functions. To test the hypothesis that the TSM response is mediated by non-specific mistranslation rather than specific Asp-->Gly misreading, we asked if streptomycin (Str), an aminoglycoside antibiotic known to promote mistranslation, can provoke a mutator phenotype. We report that Str induces a strong mutator phenotype in cells bearing certain alleles of rpsL, the gene encoding S12, an essential component of the ribosomal 30 S subunit. The phenotype is strikingly similar to that observed in mutA cells in its mutational specificity, as well as in its requirement for RecABC-mediated homologous recombination functions. Expression of Str-inducible mutator phenotype correlates with mistranslation efficiency in response to Str. Thus, mistranslation in general is able to induce the TSM response. The Str-inducible mutator phenotype described here defines a new functional class of rpsL alleles, and raises interesting questions on the mechanism of action of Str, and on bacterial response to antibiotic stress.     

Fertility of triploid hybrids of Prussian carp (<Emphasis Type="Italic">Carassius gibelio</Emphasis>) with common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Fertility of backcross triploid hybrids containing one genome of Prussian carp and two genomes of common carp is investigated. The females of hybrids of Prussian carp and common carp (Prussian × common carp) are prolific and produce diploid gametes. Since males of such hybrids are sterile, their reproduction is realized by means of induced gynogenesis. Triploid progeny is obtained by backcrossing female Prussian × common carp with carp males. Among triploids obtained from hybrids F₁ and among hybrids of the first gynogenetic generation, there were no prolific specimens. However, in reproduction of diploid hybrids by means of gynogenesis during six generations, the female fertility in the backcross progeny is restored. From backcross triploid females (daughters of Prussian × common carp of the sixth gynogenetic generation), a viable triploid gynogenetic progeny and a tetraploid backcross (by carp) progeny are obtained. The obtained data may be considered as the experimental proof of the hypothesis of reticular speciation.     

Estimation of the biological age in females of the taiga tick (Ixodes persulcatus: Ixodidae) by changing of fat resources in the organism

The method of estimation of the biological age in hungry tick females by revealing of the degree of lipid inclusions in the cells of the midgut and of the fat body is assumed. In order to estimate the degree of fat reserves in hungry females, live ticks were dissected and fragments of their internal organs were vitally stained with the saturated solution of Sudan III in 70% ethanol. Three age-specific groups were established, including (I) young females whose intestines and fat body were filled with fat inclusions; (II) mature females whose fat reserves were partially expended; and (III) old females possessing solitary fat inclusions in their midgut and fat body.     

Interaction of D-Ala2-[Tyr-3,5-3H]enkephalin(5-D-Leu) with opiate receptors of rat brain

Some kinetic features of D-Ala2-[Tyr-3.5-3H]enkephalin (5-D-Leu) binding to opiate receptors of rat brain were studied. It was shown that the Leu-enkephalin D analog interacts with the high and low affinity binding sites of opiate receptors, the equilibrium constants being equal to 0.71 and 8.4 nM, respectively. The rate constant for the label association with the high affinity binding sites in 2 . 10(8) M-1 min-1; those for the label dissociation from the opiate receptor binding sites with high and low affinities are 7.2 . 10(-3) and 0.16 min-1, respectively. Hence, the half-life time of these complexes is 95.7 and 4.3 min, respectively. Na+, K+ and Li+ markedly decrease the specific finding of the label, while Mg2+, Mn2+ and Ca2+ at the concentrations studied markedly increase its specific binding. It is concluded that the Leu-enkephalin D-analog under study acts as a morphine agonist and reveals a much higher affinity for rat brain opiate receptors than does Leu- or Met-enkephalin. This makes it a useful tool for study of the enkephalin reception under normal and pathological conditions.