The crystal structure of an odorant binding protein from Anopheles gambiae: evidence for a common ligand release mechanism

The Anopheles gambiae mosquito is the main vector of malaria transmission in sub-Saharan Africa. We present here a 1.5A crystal structure of AgamOBP1, an odorant binding protein (OBP) from the A. gambiae mosquito. The protein crystallized as a dimer with a unique binding pocket consisting of a continuous tunnel running through both subunits of the dimer and occupied by a PEG molecule. We demonstrate that AgamOBP1 undergoes a pH dependent conformational change that is associated with reduced ligand binding. A predominance of acid-labile hydrogen bonds involving the C-terminal loop suggests a mechanism in which a drop in pH causes C-terminal loop to open, leaving the binding tunnel solvent exposed, thereby lowering binding affinity for ligand. Because proteins from two distantly related insects also undergo a pH dependent conformational change involving the C-terminus that is associated with reduced ligand affinity, our results suggest a common mechanism for OBP activity.     

Crystal structure of Leishmania major peroxidase and characterization of the compound i tryptophan radical

The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX). One common feature is a key Trp residue, Trp(208) in LmP and Trp(191) in CCP, that is situated adjacent to the proximal His heme ligand in CCP, APX, and LmP. In CCP, Trp(191) forms a stable cationic radical after reaction with H(2)O(2) to form Compound I; in APX, the radical is located on the porphyrin ring. In order to clarify the role of Trp(208) in LmP and to further probe peroxidase structure-function relationships, we have determined the crystal structure of LmP and have studied the role of Trp(208) using electron paramagnetic resonance spectroscopy (EPR), mutagenesis, and enzyme kinetics. Both CCP and LmP have an extended section of β structure near Trp(191) and Trp(208), respectively, which is absent in APX. This region provides stability to the Trp(191) radical in CCP. EPR of LmP Compound I exhibits an intense and stable signal similar to CCP Compound I. In the LmP W208F mutant, this signal disappears, indicating that Trp(208) forms a stable cationic radical. In LmP conversion of the Cys(197) to Thr significantly weakens the Compound I EPR signal and dramatically lowers enzyme activity. These results further support the view that modulation of the local electrostatic environment controls the stability of the Trp radical in peroxidases. Our results also suggest that the biological role of LmP is to function as a cytochrome c peroxidase.     

Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.     

Cation-induced stabilization of the engineered cation-binding loop in cytochrome c peroxidase (CcP)

We have previously shown that the K(+) site found in the proximal heme pocket of ascorbate peroxidase (APX) could be successfully engineered into the closely homologous cytochrome c peroxidase (CcP) [Bonagura et al., (1996) Biochemistry 35, 6107-6115; Bonagura et al. (1999) Biochemistry 38, 5538-5545]. In addition, specificity could be switched to binding Ca(2+) as found in other peroxidases [Bonagura et al. (1999) J. Biol. Chem. 274, 37827-37833]. The introduction of a proximal cation-binding site also promotes conversion of the Trp191 containing cation-binding loop from a "closed" to an "open" conformer. In the present study we have changed a crucial hinge residue of the cation-binding loop, Asn195, to Pro which stabilizes the loop, albeit, only in the presence of bound K(+). The crystal structure of this mutant, N195PK2, has been refined to 1.9 A. As predicted, introduction of this crucial hinge residue stabilizes the cation-binding loop in the presence of the bound K(+). As in earlier work, the characteristic EPR signal of Trp191 cation radical becomes progressively weaker with increasing [K(+)] and the lifetime of the Trp191 radical also has been considerably shortened in this mutant. This mutant CcP exhibits reduced enzyme activity, which could be titrated to lower levels with increasing [K(+)] when horse heart cytochrome c is the substrate. However, with yeast cytochrome c as the substrate, the mutant was as active as wild-type at low ionic strength, but 40-fold lower at high ionic strength. We attribute this difference to a change in the rate-limiting step as a function of ionic strength when yeast cytochrome c is the substrate.     

The Steady State and Time-resolved Fluorescence Studies on the Lysozyme-Ligand Interaction

The conformational change of hen egg-white lysozyme (EC 3.2.1.17) induced by the interaction with tri-N-acetyl-D-glucosamine were investigated by steady state and time-resolved fluorescence spectroscopy. To identify more clearly the conformation of hen egg-white lysozyme interacting with the ligand, the fluorescence decay kinetics of the lysozyme and its complex with the ligand were precisely measured at their full spectral regions. The spectral analysis based on the time-resolved studies showed that the binding of the ligand affected not only the Trp62 directly linked to the ligand but its influence was extended to the vicinity of Trp108 and further to the hydrophobic matrix box region. Near the binding site, the intramolecular distance between Trp108 and Glu35 was expanded or contracted depending on the pH of the buffer solution. On the other hand, the interaction of Trp28 and/or Trp111 with their surroundings was reduced by restriction of fluctuational motions at the hydrophobic matrix box region.     

19F NMR study of the leucine-specific binding protein of Escherichia coli: mutagenesis and assignment of the 5-fluorotryptophan-labeled residues

The Escherichia coli L-leucine receptor is an aqueous protein and the first component in the distinct transport pathway for hydrophobic amino acids. L-leucine binding induces a conformational change, which enables the receptor to dock to the membrane components. To investigate the ligand-induced conformational change and binding properties of this protein, we used (19)F NMR to probe the four tryptophan residues located in the two lobes of the protein. The four tryptophan residues were labeled with 5-fluorotryptophan and assigned by site-directed mutagenesis. The (19)F NMR spectra of the partially ligand free proteins show broadened peaks which sharpen when L-leucine is bound, showing that the labeled wild-type protein and mutants are functional. The titration of L-phenylalanine into the 5-fluorotryptophan labeled wild-type protein shows the presence of closed and open conformers. Urea-induced denaturation studies support the NMR results that the wild-type protein binds L-phenylalanine in a different manner to L-leucine. Our studies showed that the tryptophan to phenylalanine mutations on structural units linked to the binding pocket produce subtle changes in the environment of Trp18 located directly in the binding cleft.     

Active site loop motion in triosephosphate isomerase: T-jump relaxation spectroscopy of thermal activation

As for many enzymes, the enzymatic pathway of triosephosphate isomerase (TIM) includes the partially rate determining motion of an active site loop (loop 6, residues 166-176), which must remain closed during chemistry but must open just before product release. The motion of this loop was monitored using laser induced temperature-jump relaxation spectroscopy at nanosecond to millisecond time resolution. Trp168 in the hinge of the mobile loop served as a fluorophore reporter in a mutant of the yeast enzyme. The opening rate was studied as a function of the concentration of glycerol 3-phosphate, a substrate surrogate. Monoexponential kinetics were observed; assuming a simple two-step ligand release mechanism involving an encounter complex intermediate, the time scales of loop opening and closing were derived. The opening rate of the loop at 25 degrees C was determined to be 2500 +/- 1000 s(-1), in remarkable agreement with solution and solid state NMR measurements. The closing rate at the same temperature was 46,700 +/- 1800 s(-1). The rates were also studied as a function of the sample temperature following the jump. Enthalpies of activation of the loop motion, DeltaH(close) and DeltaH(open), were estimated to be 13.8 and 14.1 kcal/mol, respectively. The enthalpy of dissociation estimated from the kinetic studies is in reasonable agreement with steady-state values. Moreover, the enthalpy was dissected, for the first time, into components associated with ion binding and with protein conformational change. The enthalpy of the release reaction appeared to have a substantial contribution from the dissociation of the ligand from the encounter complex, found to be endothermic at 6 kcal/mol. In contrast, the population ratio of the open to closed loop conformations is found to favor the closed conformation but to be substantially less temperature dependent than the release step. Preliminary data of other ligands show that G3P behavior resembles that of the substrate but differs from 2-phosphoglycolate, a tight binding inhibitor, and phosphate. This study represents one of the first detailed comparisons between NMR and fluorescence based probes of protein motion and results in good agreement between the methods. The data in aggregate support a model in which the rate of the loop opening for TIM is dependent on the ligand and results in opening rates in the presence of the product that are comparable to enzymatic throughput, kcat.     

Structural studies of the streptavidin binding loop.

The streptavidin-biotin complex provides the basis for many important biotechnological applications and is an interesting model system for studying high-affinity protein-ligand interactions. We report here crystallographic studies elucidating the conformation of the flexible binding loop of streptavidin (residues 45 to 52) in the unbound and bound forms. The crystal structures of unbound streptavidin have been determined in two monoclinic crystal forms. The binding loop generally adopts an open conformation in the unbound species. In one subunit of one crystal form, the flexible loop adopts the closed conformation and an analysis of packing interactions suggests that protein-protein contacts stabilize the closed loop conformation. In the other crystal form all loops adopt an open conformation. Co-crystallization of streptavidin and biotin resulted in two additional, different crystal forms, with ligand bound in all four binding sites of the first crystal form and biotin bound in only two subunits in a second. The major change associated with binding of biotin is the closure of the surface loop incorporating residues 45 to 52. Residues 49 to 52 display a 3(10) helical conformation in unbound subunits of our structures as opposed to the disordered loops observed in other structure determinations of streptavidin. In addition, the open conformation is stabilized by a beta-sheet hydrogen bond between residues 45 and 52, which cannot occur in the closed conformation. The 3(10) helix is observed in nearly all unbound subunits of both the co-crystallized and ligand-free structures. An analysis of the temperature factors of the binding loop regions suggests that the mobility of the closed loops in the complexed structures is lower than in the open loops of the ligand-free structures. The two biotin bound subunits in the tetramer found in the MONO-b1 crystal form are those that contribute Trp 120 across their respective binding pockets, suggesting a structural link between these binding sites in the tetramer. However, there are no obvious signatures of binding site communication observed upon ligand binding, such as quaternary structure changes or shifts in the region of Trp 120. These studies demonstrate that while crystallographic packing interactions can stabilize both the open and closed forms of the flexible loop, in their absence the loop is open in the unbound state and closed in the presence of biotin. If present in solution, the helical structure in the open loop conformation could moderate the entropic penalty associated with biotin binding by contributing an order-to-disorder component to the loop closure.     

Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.     

Interdependence of backbone flexibility,residue conservation,and enzyme function: a case study on beta1,4-galactosyltransferase-I

Beta1,4-galactosyltransferase-I (beta4Gal-T1) catalyzes the transfer of a galactose from UDP-galactose to N-acetylglucosamine. A recent crystal structure determination of the substrate-bound enzyme reveals a large conformational change, which creates binding sites for the oligosaccharide and alpha-lactalbumin, when compared to the ligand-free structure. The conformational changes take place in a 21-residue-long loop (I345-H365) and in a smaller loop containing a tryptophan residue (W314) flanked by glycines (Y311-G316; Trp loop). A series of molecular dynamics simulations carried out with an implicit solvent model and with explicit water successfully identify flexibility in the two loops and in another interacting loop. These observations are confirmed by limited proteolysis experiments that reveal an intrinsic flexibility of the long loop. The multiple simulation runs starting with the substrate-free structure show that the long loop moves toward its conformation in the ligand-bound structure; however, it gets stabilized in an intermediate position. The Trp loop moves in the opposite direction to that of the long loop, making contacts with residues in the long loop. Remarkably, when the Trp loop is restrained in its starting conformation, no large conformational change takes place in the long loop, indicating residue communication of flexibility. Sequence and structural analysis of the beta4Gal-T1 family with 37 known sequences reveals that in contrast to the unconserved long loop, which undergoes a much larger conformational change, the Trp loop including the glycines is highly conserved. These observations lead us to propose a new functional mechanism that may be conserved by evolution to perform a variety of functions.     

Kinetic mechanism of ligand binding in human ileal bile acid binding protein as determined by stopped-flow fluorescence analysis

Cooperative ligand binding to human ileal bile acid binding protein (I-BABP) was studied using the stopped-flow fluorescence technique. The kinetic data obtained for wild-type protein are in agreement with a four-step mechanism where after a fast conformational change on the millisecond time scale, the ligands bind in a sequential manner, followed by another, slow conformational change on the time scale of seconds. This last step is more pronounced in the case of glycocholate (GCA), the bile salt that binds with high positive cooperativity and is absent in mutant I-BABP proteins that lack positive cooperativity in their bile salt binding. These results suggest that positive cooperativity in human I-BABP is related to a slow conformational change of the protein, which occurs after the second binding step. Analogous to that in the intestinal fatty acid binding protein (I-FABP), we hypothesize that ligand binding in I-BABP is linked to a disorder-order transition between an open and a closed form of the protein.     

Conversion of the proximal histidine ligand to glutamine restores activity to an inactive mutant of cytochrome c peroxidase.

Using site-directed mutagenesis, a double mutant in yeast cytochrome c peroxidase (CCP) has been constructed where the proximal ligand, His175, has been converted to glutamine and the neighboring Trp191 has been converted to phenylalanine. The refined 2.4-A crystal structure of the double mutant shows that the Gln175 side chain is within coordination distance of the heme iron atom and that Phe191 occupies the same position as Trp191 in the native enzyme with very little rearrangement outside the immediate vicinity of the mutations. Consistent with earlier work, we find that the single mutant, His175-->Gln, is fully active under steady state assay conditions and that as reported earlier (Mauro et al., 1988), the Trp191-->Phe mutant exhibits only < 0.05% activity. However, the double mutant, His175-->Gln/Phe191-->Phe, exhibits 20% wild type activity. Since it is known that the Trp191-->Phe mutant is inactive because it can no longer transfer electrons from ferrocytochrome c, changing the nature of the proximal ligand is able to restore this activity. These results raise interesting questions regarding the mechanism of interprotein electron transfer reactions.     

Structural and mutational studies on an aldo‐keto reductase AKR5C3 from Gluconobacter oxydans

An aldo‐keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α‐ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3^‐D53A) in complex with NADPH are presented herein. Structure comparison and site‐directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain‐of‐function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH‐binding pocket. The AKR5C3^‐W30A and AKR5C3^‐W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3^‐W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate‐binding affinity by improving hydrophobicity in the substrate‐binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme.     

Selected-fit versus induced-fit protein binding: kinetic differences and mutational analysis

The binding of a ligand molecule to a protein is often accompanied by conformational changes of the protein. A central question is whether the ligand induces the conformational change (induced-fit), or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of ground and excited states of the protein (selected-fit). We consider here the binding kinetics in a simple four-state model of ligand-protein binding. In this model, the protein has two conformations, which can both bind the ligand. The first conformation is the ground state of the protein when the ligand is off, and the second conformation is the ground state when the ligand is bound. The induced-fit mechanism corresponds to ligand binding in the unbound ground state, and the selected-fit mechanism to ligand binding in the excited state. We find a simple, characteristic difference between the on- and off-rates in the two mechanisms if the conformational relaxation into the ground states is fast. In the case of selected-fit binding, the on-rate depends on the conformational equilibrium constant, whereas the off-rate is independent. In the case of induced-fit binding, in contrast, the off-rate depends on the conformational equilibrium, while the on-rate is independent. Whether a protein binds a ligand via selected-fit or induced-fit thus may be revealed by mutations far from the protein's binding pocket, or other "perturbations" that only affect the conformational equilibrium. In the case of selected-fit, such mutations will only change the on-rate, and in the case of induced-fit, only the off-rate.     

Active site and loop 4 movements within human glycolate oxidase: implications for substrate specificity and drug design

Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1,2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most alpha-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with alpha-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.     

A mobile tryptophan is the intrinsic charge transfer donor in a flavoenzyme essential for nikkomycin antibiotic biosynthesis

The flavoenzyme nikD is required for the biosynthesis of nikkomycin antibiotics. NikD exhibits an unusual long wavelength absorption band attributed to a charge transfer complex of FAD with an unknown charge transfer donor. NikD crystals contain an endogenous active site ligand. At least four different compounds are detected in nikD extracts, including variable amounts of two ADP derivatives that bind to the enzyme's dinucleotide binding motif in competition with FAD, picolinate (0.07 mol/mol of nikD) and an unknown picolinate-like compound. Picolinate, the product of the physiological catalytic reaction, matches the properties deduced for the active site ligand in nikD crystals. The charge transfer band is eliminated upon mixing nikD with excess picolinate but not by a reversible unfolding procedure that removes the picolinate-like compound, ruling out both compounds as the intrinsic charge transfer donor. Mutation of Trp355 to Phe eliminates the charge transfer band, accompanied by a 30-fold decrease in substrate binding affinity. The results provide definitive evidence for Trp355 as the intrinsic charge transfer donor. The indole ring of Trp355 is coplanar with or perpendicular to the flavin ring in "open" or "closed" crystalline forms of nikD, respectively. Importantly, a coplanar configuration is required for charge transfer interaction. Absorption in the long wavelength region therefore constitutes a valuable probe for monitoring conformational changes in solution that are likely to be important in nikD catalysis.     

Dynamics of the WPD loop of the Yersinia protein tyrosine phosphatase

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia spp., causing gastrointestinal diseases and the plague. Like eukaryotic PTPases, YopH catalyzes the hydrolysis of the phosphate moiety of phosphotyrosine within a highly conserved binding pocket, which is also characterized by the closure of the so-called "WPD loop" upon ligand binding. In this study, we investigate the conformational changes and dynamics of the WPD loop by molecular dynamics simulations. Consistent with experimental observations, our simulations show that the WPD loop of YopH is intrinsically flexible and fluctuates between the open and closed conformation with a frequency of approximately 4 ns for the apo, native protein. The region of helix alpha4 spanning loop 384-392, which has been revealed experimentally as a second substrate-binding site in YopH, is found to be highly associated with the WPD loop, stabilizing it in the closed, active conformation, and providing a structural basis for the cooperation of the second-substrate binding site in substrate recognition. Loop L4 (residues 323-327) is shown to be involved in a parallel, correlated motion mode with the WPD loop that contributes the stabilization of a more extended open conformation. In addition, we have simulated the loop reopening in the ligand-bound protein complex by applying the locally enhanced sampling method. Finally, the dynamic behavior of the WPD loop for the C403S mutant differs from the wild-type YopH remarkably. These results shed light on the role of the WPD loop in PTPase-mediated catalysis, and are useful in structure-based design for novel, selective YopH inhibitors as antibacterial drugs.     

The effects of an engineered cation site on the structure, activity, and EPR properties of cytochrome c peroxidase

Earlier work [Bonagura et al. (1996) Biochemistry 35, 6107] showed that the K+ site found in the proximal pocket of ascorbate peroxidase (APX) could be engineered into cytochrome c peroxidase (CCP). Binding of K+ at the engineered site results in a loss in activity and destabilization of the CCP compound I Trp191 cationic radical owing to long-range electrostatic effects. The engineered CCP mutant crystal structure has been refined to 1.5 A using data obtained at cryogenic temperatures which provides a more detailed basis for comparison with the naturally occurring K+ site in APX. The characteristic EPR signal associated with the Trp191 radical becomes progressively weaker as K+ is added, which correlates well with the loss in enzyme activity as [K+] is increased. These results coupled with stopped-flow studies support our earlier conclusions that the loss in activity and EPR signal is due to destabilization of the Trp191 cationic radical.