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R K Crouch

Publications and source records attributed to R K Crouch.

At least 37 records · Page 2Linked to original sources
PubMed1999

The proton release group of bacteriorhodopsin controls the rate of the final step of its photocycle at low pH.

The factors determining the pH dependence of the formation and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle were investigated in the wild-type (WT) pigment and in the mutants of Glu-194 and Glu-204, key residues of the proton release group (PRG) in bR. We have found that in the WT the rate constant of O --> bR transition decreases 30-fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O slows the rise and decay of the O intermediate in the WT at pH 3.5 by a factor of 5.5. We suggest that the rate of the O --> bR transition (which reflects the rate of deprotonation of the primary proton acceptor Asp-85) at low pH is controlled by the deprotonation of the PRG. To test this hypothesis, we studied the E194D mutant. We show that the pKa of the PRG in the ground state of the E194D mutant, when Asp-85 is protonated, is increased by 1.2 pK units compared to that of the WT. We found a similar increase in the pKa of the rate constant of the O --> bR transition in E194D. This provides further evidence that the rate of the O --> bR transition is controlled by the PRG. In a further test, the E194Q mutation, which disables the PRG and slows proton release, almost completely eliminates the pH dependence of O decay at pHs below 6. A second phenomenon we investigated was that in the WT at neutral and alkaline pH the fraction of the O intermediate decreases with pKa 7.5. A similar pH dependence is observed in the mutants in which the PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction of the O intermediate with pKa ca. 7.5 is not controlled by the PRG. We propose that the group with pKa 7.5 is Asp-96. The slowing of the reprotonation of Asp-96 at high pH is the cause of the decrease in the rate of the N --> O transition, leading to the decrease in the fraction of O.

Aspartic Acid↗
PubMed1999

Two groups control light-induced Schiff base deprotonation and the proton affinity of Asp85 in the Arg82 his mutant of bacteriorhodopsin.

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 micros to 75 micros with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.

Absorption↗
PubMed1999

Categorizing reactivity of bacteriorhodopsin cysteine mutants crosslinking to 4-bromoretinal.

The structure of bacteriorhodopsin (bR) has been probed by a large number of experimental methods. In earlier work distance constraints measured from the 1BRD Brookhaven structure (1, 2) were used to guide site-directed mutagenesis/affinity labeling experiments (3-5). In the present study we report on the use of limited molecular dynamics (MD) investigations of the same bR/affinity label system. We show here that the chiral center introduced when 4-bromo-all-trans retinal is synthesized produces variable impact on potential crosslinking. Our MD analysis suggests the following ranking of binding site mutants in order of reactivity: R118C > S118C >> S121C > R141C >> S141C >>> R121C, R138C, S138C. Chirality appears to have limited effect for the M118C mutants but shows more dramatic impact for the T121C and S141C mutants. These results are in excellent agreement with the experimental observations and offer encouragement that MD can be a useful component of experimental design with considerable predictive power.

Bacterial Proteins↗
PubMed1999

Occupancy of the chromophore binding site of opsin activates visual transduction in rod photoreceptors.

The retinal analogue beta-ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a steady state, and then were exposed to beta-ionone. Our experiments show that in bleach-adapted rods beta-ionone causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in bleach-adapted rods beta-ionone activates phototransduction in the dark. Control experiments showed no effect of beta-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of beta-ionone with the free opsin produced by bleaching. We speculate that beta-ionone binds specifically in the chromophore pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin. We show that binding of beta-ionone to rod opsin produces effects opposite to its previously described effects on cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors.

Algorithms↗
PubMed1999

Studies on pyrylretinal analogues of bacteriorhodopsin.

The retinal analogues 3-methyl-5-(1-pyryl)-2E,4E-pentadienal (1) and 3,7-dimethyl-9-(1-pyryl)-2E,4E,6E,8E-nonatetr aenal (2), which contain the tetra aromatic pyryl system, have been synthesized and characterized in order to examine the effect of the extended ring system on the binding capabilities and the function of bacteriorhodopsin (bR). The two bR mutants, E194Q and E204Q, known to have distinct proton-pumping patterns, were also examined so that the effect of the bulky ring system on the proton-pumping mechanism could be studied. Both retinals formed pigments with all three bacterioopsins, and these pigments were found to have absorption maxima in the range 498-516 nm. All the analogue pigments showed activity as proton pumps. The pigment formed from wild-type apoprotein bR with 1 (with the shortened polyene side chain) showed an M intermediate at 400 nm and exhibited fast proton release followed by proton uptake. Extending the polyene side chain to the length identical with retinal, analogue 2 with wild-type apoprotein gave a pigment that shows M and O intermediates at 435 nm and 650 nm, respectively. This pigment shows both fast and slow proton release at pH 7, suggesting that the pKa of the proton release group (in the M-state) is higher in this pigment compared to native bR. Hydrogen azide ions were found to accelerate the rise and decay of the O intermediate at neutral pH in pyryl 2 pigment. The pigments formed between 2 and E194Q and E204Q showed proton-pumping behavior similar to pigments formed with the native retinal, suggesting that the size of the chromophore ring does not alter the protein conformation at these sites.

Bacteriorhodopsins↗
PubMed1998

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells1.

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of the salamander red and blue cone visual pigments.

PURPOSE: Salamander retinas are known to contain at least three cone pigments and two rod pigments. The purpose of this study was to clone and characterize the visual pigments from salamander cones. METHODS: cDNA fragments of cone pigments were amplified from a salamander retina cDNA library by PCR using a pair of primers with consensus for visual pigments. These fragments were cloned and used as probes for library-screening. The full-length cDNAs were isolated from the retinal library using the cloned PCR products as probes. DNA sequences were determined by the dideoxynucleotide chain termination method. RESULTS: Two pigment cDNAs were cloned and sequenced from the salamander library. The global GenBank search showed that they do not match any existing sequences but have significant sequence similarity to visual pigments. One of the pigment cDNAs showed a high sequence homology with red cone pigments from other species and thus, was designated as a red cone opsin. The other pigment was designated as a blue cone opsin as it is most homologous to the chicken and goldfish blue cone pigments. Both cDNAs contain a full-length coding region encoding 365 amino acids in the red and 363 amino acids in the blue cone pigment. Hydropathy analysis predicted that both pigments could form seven hydrophobic transmembrane helices. Both pigments retain the key amino acid residues critical for maintaining the structure and function of opsins and have similar G-protein interaction sequences which differ from that of rod opsin. Phylogenetic analysis indicates that the red opsin belongs to the L group and the blue opsin belongs to the M1 group of visual pigments. CONCLUSIONS: The salamander red and blue cone pigments share high sequence homology with the cone pigments of other species.

Amino Acid Sequence↗
PubMed1998

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.

Amino Acid Sequence↗
PubMed1998

Identification of tryptophan oxidation products in bovine alpha-crystallin.

Oxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal-catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of alpha-crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine alpha-crystallin sequence. After oxidative exposure, alpha-crystallin was digested with trypsin, and the resulting peptides were fractionated by reverse-phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of alphaA- and tryptophan 60 of alphaB-crystallin were each converted into hydroxytryptophans (HTRP), N-formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of alphaB-crystallin. Oxidation of methionine 1 of alphaA- and methionine 1 and 68 of alphaB-crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact alpha-crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.

5-Hydroxytryptophan↗
PubMed1998

Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle.

Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

Animals↗
PubMed1998

Radiolysis-induced oxidation of bovine alpha-crystallin.

Radiolysis of water by ionizing radiation results in the production of pure hydroxyl radicals. This technique, combined with analysis by tandem mass spectrometry (MS/MS), has been used to study the effect of hydroxyl radicals on the intact bovine alpha-crystallin protein. After exposure to gamma-irradiation, the oxidized alpha-crystallin was digested with trypsin and the resulting peptides were fractionated by reverse-phase HPLC. The isolated fractions were analyzed by matrix-assisted laser desorption ionization and by MS/MS to determine the locations and identities of the modifications. Structural analysis revealed that methionine 1 of alpha A- and alpha B-crystallin and methionine 68 of alpha B-crystallin were oxidized to methionine sulfoxide. Hydroxytryptophan was formed from each tryptophan residue in alpha-crystallin, although only tryptophan 9 of alpha A-crystallin was converted into N-formylkynurenine. This study has, for the first time, identified the sites of modification and the structures produced in the intact alpha-crystallin protein by exposure to hydroxyl radicals. By determining the consequences of in vitro exposure of alpha-crystallin to pure hydroxyl radicals, the in vivo contribution of this reactive oxygen species to the overall oxidative stress of the lens will be achieved from the identification of the modifications to alpha-crystallin purified from intact human lenses.

Amino Acid Sequence↗
PubMed1997

Glutamate-194 to cysteine mutation inhibits fast light-induced proton release in bacteriorhodopsin.

Substitution of glutamic acid-194, a residue on the extracellular surface of bacteriorhodopsin, with a cysteine inhibits the fast light-induced proton release that normally is coupled with the deprotonation of the Schiff base during the L to M transition. Proton release in this mutant occurs at the very end of the photocycle and coincides with deprotonation of the primary proton acceptor, Asp-85, during the O to bR transition. the E194C mutation also results in a slowing down of the photocycle by about 1 order of magnitude as compared to the wild type and produces a strong effect on the pH dependence of dark adaptation that is interpreted as a drastic reduction or elimination of the coupling between the primary proton acceptor Asp-85 and the proton release group. These data indicate that Glu-194 is a critical component of the proton release complex in bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1997

Proton uptake and release are rate-limiting steps in the photocycle of the bacteriorhodopsin mutant E204Q.

In the absence of the putative proton release group, E204, the second half of the photocycle of the E204Q mutant of bacteriorhodopsin is slowed down more than 10-fold compared to the wild type. The effects of pH and D2O on the M decay and O formation rates in E204Q suggest that proton uptake occurs concurrently with the N <--> O transition, possibly coupled with the thermal reisomerization of the retinal. Hence, one of the rate-limiting steps in the slow E204Q photocycle is proton uptake from the outside medium, coincident with the decay of the slow component of M (the N <--> O transition). The second rate-limiting step is the long lifetime of decay of the O state, due to a high activation barrier for the deprotonation of D85 in the O --> bR step of the E204Q photocycle. Addition of the weakly acidic anions azide, cyanate, or formate accelerates the decay of the O intermediate, and restores the total photocycling time to that observed in the wild-type pigment, by accelerating the deprotonation of D85. We also find that azide similarly accelerates the decay of O in the wild type under conditions in which E204 does not deprotonate during the photocycle (pH < 6). It has previously been shown that azide and other weak acids can influence proton transfers in the cytoplasmic half of the protein [Tittor, J., Soell, C., Oesterhelt, D., Butt, H.-J., & Bamberg, E. (1989) EMBO J. 8, 3477-3482]; we suggest that these weak acids can affect proton transfers in the extracellular half of the protein as well.

Azides↗
PubMed1997

Mutation of a surface residue, lysine-129, reverses the order of proton release and uptake in bacteriorhodopsin; guanidine hydrochloride restores it.

K129 is a residue located in the extracellular loop connecting transmembrane helices D and E of bacteriorhodopsin. Replacement of K129 with a histidine alters the pKa's of two key residues in the proton transport pathway, D85, and the proton release group (probably E204); the resulting pigment has properties that differ markedly from the wild type. 1) In the unphotolyzed state of the K129H mutant, the pKa of D85 is 5.1 +/- 0.1 in 150 mM KCl (compared to approximately 2.6 in the wild-type bacteriorhodopsin), whereas the unphotolyzed-state pKa of E204 decreases to 8.1 +/- 0.1 (from approximately 9.5 in the wild-type pigment). 2) The pKa of E204 in the M state is 7.0 +/- 0.1 in K129H, compared to approximately 5.8 in the wild-type pigment. 3) As a result of the change in the pKa of E204 in M, the order of light-induced proton release and uptake exhibits a dependence on pH in K129H differing from that of the wild type: at neutral pH and moderate salt concentrations (150 mM KCl), light-induced proton uptake precedes proton release, whereas it follows proton release at higher pH. This pumping behavior is similar to that seen in a related bacterial rhodopsin, archaerhodopsin-1, which has a histidine in the position analogous to K129. 4) At alkaline pH, a substantial fraction of all-trans K129H pigment (approximately 30%) undergoes a conversion into a shorter wavelength species, P480, with pKa approximately 8.1, close to the pKa of E204. 5) Guanidine hydrochloride lowers the pKa's of D85 and E204 in the ground state and the pKa of E204 in the M intermediate, and restores the normal order of proton release before uptake at neutral pH. 6) In the K129H mutant the coupling between D85 and E204 is weaker than in wild-type bacteriorhodopsin. In the unphotolyzed pigment, the change in the pKa's of either residue when the other changes its protonation state is only 1.5 units compared to 4.9 units in wild-type bacteriorhodopsin. In the M state of photolyzed K129H pigment, the corresponding change is 1 unit, compared to 3.7 units in the wild-type pigment. We suggest that K129 may be involved in stabilizing the hydrogen bonding network that couples E204 and D85. Substitution of K129 with a histidine residue causes structural changes that alter this coupling and affect the pKa's of E204 and D85.

Bacteriorhodopsins↗
PubMed1997

Formation of free radicals and protein mixed disulfides in rat red cells exposed to dapsone hydroxylamine.

The hemolytic activity of dapsone is well known to reside in its N-hydroxylamine metabolites. Addition of dapsone hydroxylamine (DDS-NOH) to red cell suspensions causes damage such that when reintroduced into the circulation of isologous rats, the injured cells are rapidly removed by the spleen. Hemolytic activity is associated with the extensive formation of disulfide-linked hemoglobin adducts on red cell membrane skeletal proteins. To determine if free radicals could be involved in this process, rat red cells were incubated with DDS-NOH in the presence of the spin trap, 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) and subjected to EPR analysis. Addition of DDS-NOH (25-50 microM) to a red cell suspension gave rise to a four-line (1:2:2:1) EPR spectrum with coupling constants identical to those of a DMPO-hydroxyl radical adduct (DMPO-OH) standard. No other radicals were detected; however, preincubation of red cells with cysteamine caused the DDS-NOH-generated DMPO-OH signal to be replaced by a cysteamine thiyl radical adduct signal. DDS-NOH-treated red cells were also found to contain ferrylhemoglobin, indicating the presence of hydrogen peroxide. Furthermore, DDS-NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confirming the presence of oxygen radicals. These data support the hypothesis that oxygen radicals are involved in the mechanism underlying dapsone-induced hemolytic anemia.

Animals↗
PubMed1997

Identification of photooxidation sites in bovine alpha-crystallin.

Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine alpha-crystallin, a major lens protein with molecular chaperone activity, were identified using tandem mass spectrometry (MS/MS). Bovine alpha-crystallin was irradiated with UV light (> 293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI) to identify the oxidized sequences. Tryptic peptides were purified by reverse-phase HPLC and oxidized peptides were sequenced by MS/MS to determine the sites of oxidation. Tryptophan fluorescence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of alpha A-crystallin and 1-11, 12-22 and 57-69 of alpha B-crystallin were determined to be oxidized by shifts of 16 D or multiples of 16 Da above the mass of the unmodified peptide. The MALDI analysis revealed single oxidation of all four sequences, which increased with increasing time of UV exposure and possible double oxidation of alpha B 12-22. The specific sites of photooxidation indicate that the N-terminal regions of alpha A- and alpha B-crystallin are exposed to an aqueous environment and are in the vicinity of tryptophan residues from neighboring subunits.

Amino Acid Sequence↗
PubMed1997

Mutation of arginine 134 to lysine alters the pK(a)s of key groups involved in proton pumping by bacteriorhodopsin.

Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light-induced Schiff-base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff-base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pK(a)s of D85 and the proton release group from acid (purple-to-blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counterion, D85, and of the proton release group. We calculate, in R134K, that the pKa of D85 is 4.6 in the unphotolyzed state, while the pKa of the proton release group is 8.0 in the unphotolyzed state but drops to approximately 5.8 in the M intermediate. The same value for the pKa of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release. The altered values of these pK(a)s relative to the corresponding values in wild-type bR suggest that D85 and the proton release group are coupled more weakly in R134K than in the wild type.

Arginine↗