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.     

Fourier transform infrared study of the halorhodopsin chloride pump

Halorhodopsin (hR) is a light-driven chloride pump located in the cell membrane of Halobacterium halobium. Fourier transform infrared difference spectroscopy has been used to study structural alterations occurring during the hR photocycle. The frequencies of peaks attributed to the retinylidene chromophore are similar to those observed in the spectra of the related protein bacteriorhodopsin (bR), indicating that in hR as in bR an all-trans----13-cis isomerization occurs during formation of the early bathoproduct. Spectral features due to protein structural alterations are also similar for the bR and hR photocycles. For example, formation of the red-shifted primary photoproducts of both hR and bR results in similar carboxyl peaks in the 1730-1745-cm-1 region. However, in contrast to bR, no further changes are observed in the carboxyl region during subsequent steps in the hR photocycle, indicating that additional carboxyl groups are not directly involved in chloride translocation. Overall, the close similarity of vibrations in hR and bR photoproduct difference spectra supports the existence of some common elements in the molecular mechanisms of energy transduction and active transport by these two proteins.     

Mechanism of a molecular valve in the halorhodopsin chloride pump

Halorhodopsin is a light-driven chloride anion pump in which the trans-->cis photoisomerization of a retinal chromophore triggers a photocycle resulting in the translocation of chloride across the plasma membrane. The mechanism of chloride transfer past the cis retinal is determined here by computing multiple pathways for this process. The calculations reveal two conditions of the valve mechanism. First, a lumen absent in the ground state structure is transiently opened by chloride passage. Second, this activated opening, which is achieved by flexible deformation of the surrounding protein, is shown to significantly raise the chloride translocation barrier between photocycles, thus preventing chloride backflow. Unlike macroscopic valve designs, the protein allows differential ion flows in the pumping and resting states that are tuned to match the physiological timescales of the cell, thus creating a "kinetic" valve.     

Roles of cytoplasmic arginine and threonine in chloride transport by the bacteriorhodopsin mutant D85T

In the light-driven anion pump halorhodopsin (HR), the residues arginine 200 and threonine 203 are involved in anion release at the cytoplasmic side of the membrane. Because of large sequence homology and great structural similarities between HR and bacteriorhodopsin (BR), it has been suggested that anion translocation by HR and by the chloride-pumping BR mutant BR-D85T occurs by the same mechanism. Consequently, the functions of the R200/T203 pair in HR should be the same as those of the corresponding pair in BR-D85T (R175/T178). We have put this hypothesis to a test by creating two mutants of BR-D85T in which R175 and T178 were replaced by glutamine and valine, respectively. Chloride transport activities were essentially the same for all three mutants, whereas chloride binding and the kinetics of parts of the photocycle were markedly affected by the replacement of T178. In contrast, the consequences of mutating R175 proved to be less significant. These findings are consistent with evidence obtained on HR and therefore support the idea that the respective mechanistic roles of the cytoplasmic arginine/threonine pairs in HR and BR-D85T are equal.     

Effects of anion binding on the deprotonation reactions of halorhodopsin

The retinal Schiff base of halorhodopsin deprotonates with a pKa of 7.4 in 0.5 M Na2SO4 in the dark. In the presence of various anions, such as chloride or nitrate, etc., the pKa is raised by up to 1.5 units. Analysis of the dependency of the pKa on anion concentration favors the model in which the anions do not bind to the positively charged Schiff base nitrogen, but to a site near it, and exert their effect on the pKa by direct (perhaps electrostatic) interaction. Adding nitrate, or one of several other anions, causes also a small blueshift in the visible absorption band of the chromophore. These effects on the pKa and the absorption band define an anion binding site in halorhodopsin, termed Site I. Chloride and bromide apparently bind in addition to another site, which is associated with a small red-shift of the absorption band and changes in the photocycle. This other anion binding site is termed Site II. Illumination of halorhodopsin samples results in the deprotonation of the Schiff base with a much lowered pKa, but at very low rates probably determined by the generation of a deprotonating photointermediate. Binding of Site I anions increases the pKa of deprotonation in the light also. The similarity of the responses of the apparent pKa in the dark and in the light to anion concentration suggests that anion binding to Site I influences deprotonation of the Schiff base similarly in the photointermediate and in the parent halorhodopsin molecule.     

Homologous overexpression of a light-driven anion pump in an archaebacterium

The retinal protein halorhodopsin (HR), a light-driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halo-opsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio-opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop-hop-cartridge was transformed into a HR-deficient strain and blueish-coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane-bound state. This allows for direct use in biophysical studies without the application of deter-gents. This was the first successful overexpression of a 7-helical transmembrane protein and may be extended to other proteins of this family.     

Picosecond events in the photochemical cycle of the light-driven chloride-pump halorhodopsin.

The early events in halorhodopsin after light excitation are studied with picosecond time resolution. Absorption and fluorescence measurements show that the electronically excited state of the incorporated retinal has a lifetime of 5 ps. Within that time a red-shifted photoproduct is formed that remains stable for at least 2 ns.     

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.     

Heterologous expression of Pharaonis halorhodopsin in Xenopus laevis oocytes and electrophysiological characterization of its light-driven Cl- pump activity

Natronomonas pharaonis halorhodopsin (pHR) is an archaeal rhodopsin functioning as an inward-directed, light-driven Cl- pump. To characterize the electrophysiological features of the Cl- pump activity of pHR, we expressed pHR in Xenopus laevis oocytes and analyzed its photoinduced Cl- pump activity using the two-electrode voltage-clamp technique. Photoinduced outward currents were observed only in the presence of Cl-, Br-, I-, NO3-, and SCN-, but not in control oocytes, indicating that photoinduced anion currents were mediated by pHR. The relationship between photoinduced Cl- current via pHR and the light intensity was linear, demonstrating that transport of Cl- is driven by a single-photon reaction and that the steady-state current is proportional to the excited pHR molecule. The current-voltage relationship for pHR-mediated photoinduced currents was also linear between -150 mV and +50 mV. The slope of the line describing the current-voltage relationship increased as the number of the excited pHR molecules was increased by the light intensity. The reversal potential (VR) for Cl- as the substrate for the anion pump activity of pHR was about -400 mV. The value for VR was independent of light intensity, meaning that the VR reflects the intrinsic value of the excited pHR molecule. The value of VR changed significantly for the R123K mutant of pHR. We also show that the Cl- pump activity of pHR can generate a substantial negative membrane potential, indicating that pHR is a very potent Cl- pump. We have also analyzed the kinetics of voltage-dependent Cl- pump activity as well as that of the photocycle. Based on these data, a kinetic model for voltage-dependent Cl- transport via pHR is presented.     

New evidence for the essential role of arginine residues in anion transport across the red blood cell membrane

2,3-Butanedione, in the dark and in the presence of borate, reacts rapidly to inactivate the sulfate equilibrium exchange across the human red cell membrane. Reactivation occurs spontaneously after the removal of borate, indicating the reaction of butanedione with essential arginine residues. The inactivation of the transport system depends on the concentration of the reagent, on the incubation time and exhibits pseudo-first-order kinetics. Chloride ions are able to protect the transport system against inactivation with the reagent. This would suggest the participation of the modified residue in the substrate binding site. When the transport system is inhibited to 50-60% by butanedione, the transporter can still bind covalently the anion transport inhibitor 2H2DIDS up to 85 +/- 12% of its total binding capacity. 3H2DIDS concentration was either 3.15, 10 or 20 microM. Modification of resealed ghosts with 50 mM butanedione under conditions where the transport system is to more than 75% inhibited, causes a reduction of only about 30% of the reversibly bound 3H2DIDS.     

Arginyl residues and anion binding sites in proteins

Summary The functions of a number of amino acid residues in proteins have been studied by chemical modification techniques and much useful information has been obtained. Methods using dicarbonyl compounds for the modification of arginine residues are the most recent to have been developed. Since their introduction about 10 years ago, they have led to the identification of a large number of enzymes and other proteins that contain arginine residues critical to biological function. These reagents are discussed in terms of their chemical reactivity and mechanisms of action and in relation to the unique chemical properties of the guanidinium group. Butanedione, phenylglyoxal and cyclohexanedione are the most commonly employed arginyl reagents, and their relative advantages are examined. A survey of the functional role of arginine residues in enzymes and other proteins is presented in which nearly 100 examples are cited. The prediction that arginine residues would be found to serve a general role as anionic binding sites in protein has obviously been validated. The genetic and physiological implications of the selection of arginine for this important function are discussed.This work was supported by Grant-in-Aid GM-15003 from the National Institutes of Health.     

A Cl and Cl NMR study of chloride binding to the erythrocyte anion transport protein

Band 3, the erythrocyte anion transport protein, mediates the one-for-one exchange of bicarbonate and chloride ions across the membrane and consequently plays an important role in respiration. Binding to the protein forms the first step in the translocation of the chloride across the membrane. ³⁵Cl and ³⁷Cl NMR relaxation measurements at various field strengths were used to study chloride binding to the protein in the presence and absence of the transport inhibitor 4,4′-dinitrostilbene-2,2′-disulfonate. Significant differences occurred in the NMR relaxation rates depending on whether the inhibitor was present or not. The results indicate that the rate of chloride association and dissociation at each external binding site occurs on a time scale of 5 μs. This implies that the transmembrane flux is not limited by the rate of chloride binding to the external chloride binding site of band 3. The rotational correlation-time of chloride bound to band 3 was found to be 20 ns with a quadrupole coupling constant of 3 MHz.     

Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.     

Chromophore/protein and chromophore/anion interactions in halorhodopsin.

Halorhodopsin (HR), the light-driven chloride transport pigment of Halobacterium halobium, was bleached and reconstituted with retinal analogues with the pi electron system interrupted at different locations (dihydroretinals). The absorption maxima of the artificial pigments formed with the dihydroretinals are found to be very similar to those of the corresponding pigments formed by reconstitution of bacteriorhodopsin (BR) and sensory rhodopsin (SR). This strongly suggests that the distribution of charges around the retinal is similar in all three bacterial rhodopsins. Comparison of the primary, and proposed secondary, structures for HR and BR reveal conserved asparagine (asp) and arginine (arg) residues, which are likely candidates for the ionizable amino acids that interact with the retinal. In a second set of experiments absorption shifts due to the binding of anions to Sites I and II in HR, reconstituted with different retinal analogues, were used to estimate the locations of these binding sites relative to the retinal. Site I is localized near the Schiff base, and Site II near the ionone ring. On the basis of these results a structural model for HR is proposed, which accounts for the spectroscopic properties of HR in terms of the three buried arg residues and two of the buried asp residues in the protein.     

On the relationship between anion binding and chloride conductance in the CFTR anion channel

Mutations at many sites within the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore region result in changes in chloride conductance. Although chloride binding in the pore – as well as interactions between concurrently bound chloride ions – are thought to be important facets of the chloride permeation mechanism, little is known about the relationship between anion binding and chloride conductance. The present work presents a comprehensive investigation of a number of anion binding properties in different pore mutants with differential effects on chloride conductance. When multiple pore mutants are compared, conductance appears best correlated with the ability of anions to bind to the pore when it is already occupied by chloride ions. In contrast, conductance was not correlated with biophysical measures of anion:anion interactions inside the pore. Although these findings suggest anion binding is required for high conductance, mutations that strengthened anion binding had very little effect on conductance, especially at high chloride concentrations, suggesting that the wild-type CFTR pore is already close to saturated with chloride ions. These results are used to support a revised model of chloride permeation in CFTR in which the overall chloride occupancy of multiple loosely-defined chloride binding sites results in high chloride conductance through the pore.     

The role of arginine residues in interleukin 1 receptor binding.

Interleukin 1 (IL-1) is a family of polypeptide cytokines that plays an essential role in modulating immune and inflammatory responses. IL-1 activity is mediated by either of two distinct proteins, IL-1 alpha or IL-1 beta, both of which bind to the same receptor found on T-lymphocytes, fibroblasts and endothelial cells (Type 1 receptor). The effect of specific chemical modification of recombinant IL-1 alpha and IL-1 beta on receptor binding was examined. Modification of the proteins with phenylglyoxal, an arginine-specific reagent, resulted in the loss of Type 1 IL-1 receptor binding activity. The stoichiometry of this modification revealed that a single arginine in either IL-1 alpha or IL-1 beta is responsible for the loss of activity. Cyanogen bromide cleavage of phenylglyoxal modified IL-1 alpha and IL-1 beta, followed by sequencing of the peptides, revealed that arginine-12 in IL-1 alpha and arginine-4 in IL-1 beta, which occupy the same topology in the respective crystallographic structures, are the target of phenylglyoxal. These results suggest that an arginine residue plays an important role in ligand-receptor interaction.     

Functional arginine residues involved in coenzyme binding by glutamate dehydrogenases.

Reaction of the NADP-dependent glutamate dehydrogenase of Neurospora with 1,2-cyclohexanedione results in a biphasic loss of enzyme activity. At the end of the rapid phase of the reaction (t1/2 = 1.5 min) the enzyme activity is diminished by approximately 60% with the simultaneous loss of 1 residue of arginine per subunit. After 60 min, the enzyme activity is completely lost with the modification of a total of 2 arginine residues per subunit. Reaction of bovine liver glutamate dehydrogenase with cyclohexanedione causes a rapid loss of approximately 45% of the enzyme activity and modification of about 1.5 residues of arginine per subunit. More prolonged treatment results in reaction of an additional 4 residues of arginine per subunit but is without further effect on the residual activity. The activity of the Neurospora enzyme is not protected by substrate, coenzyme, or a combination of both; however, the activity of the bovine enzyme is partially protected by high levels of NAD or NADP. Although the Km for alpha-ketoglutarate is unchanged by a limited modification of either enzyme with cyclohexanedione, the Km for coenzyme is increased about 2-fold for the Neurospora enzyme and about 1.5-fold for the bovine enzyme. The Ki of the Neurospora dehydrogenase for the competitive inhibitor 2'-monophosphoadenosine-5'-diphosphoribose is unchanged by the enzyme modification, but nicotinamide mononucleotide, a competitive inhibitor for the native Neurospora enzyme, does not inhibit the glutamate dehydrogenase with 1 modified arginine residue. This finding implies that the modified arginine is at or near the nicotinamide binding iste of the enzyme.     

Inhibition of chloride binding to the anion transport site by diethylpyrocarbonate modification of band 3

Summary The line widths of³⁵Cl^– nuclear magnetic resonances were used to measure chloride binding by Band 3. Since this procedure related directly to binding, the data obtained may be interpreted more unequivocally than affinities derived from kinetic data which could be related to either translocation or binding. Chloride binding to the active sites in Band 3 was assessed from that portion of the total line width which was sensitive to 4,4-dinitrostilbene-2,2-disulfonic acid. These sites appeared to be completely inhibited by treatment of erythrocyte membranes with diethylpyrocarbonate. This result is consistent with our previous observation that this reagent inhibits anion transport in resealed erythrocyte ghosts (Izuhara, Okubo & Hamasaki, 1989,Biochemistry 28:4725–4728). Hydroxylamine could not reverse the diethylpyrocarbonate inhibition of chloride binding to Band 3. The pH-dependence of diethylpyrocarbonate reactivity suggests that the modified residues may be those of histidine.