Role of axial ligands in the reactivity of Mn peroxidase from Phanerochaete chrysosporium.

Site-directed mutagenesis was performed on Mn peroxidase (MnP) from the white-rot fungus Phanerochaete chrysosporium to investigate the role of the axial ligand hydrogen-bonding network on heme reactivity. D242 is hydrogen bonded to the proximal His of MnP; in other peroxidases, this conserved Asp, in turn, is hydrogen bonded to a Trp. In MnP and other fungal peroxidases, the Trp is replaced by a Phe (F190). Both residues are thought to have a direct influence on the electronic environment of the catalytic center. To study only the active mutants at D242 and F190, we used degenerate oligonucleotides allowing us to screen all 19 possible amino acid mutants at these positions. Two mutants at D242 passed our screen, D242E and D242S. Both mutations impaired only the functioning of compound II. The reactions of the ferric enzyme with H(2)O(2) were unaffected by the mutations, as were the reactions of compound I with reducing substrates. The D242S and D242E mutations reduced the first-order rate constant for the reaction of MnP compound II with chelated Mn(2+) from 233 s(-1) (wild type) to 154 s(-1) and 107 s(-1), respectively. Three F190 mutants passed our screen, F190V, F190L, and F190W. Similar to mutants at D242, these mutants largely affected the function of compound II. The F190V mutation increased the first-order rate constant for the reduction of compound II by chelated Mn(2+) to 320 s(-1). The F190L mutation decreased this rate to 137 s(-1). The F190W mutant was not very stable, but at pH 6.0, this mutation decreased the rate of compound II reduction by Mn(2+) from 140 s(-1) in the wild type to 36 s(-1). There was no indication that the F190W mutant was capable of forming a protein-centered Trp cation radical. All the mutations altered the midpoint potential of the Fe(3+)/Fe(2+) couple of the enzyme, as calculated from cyclic voltammagrams of the proteins. The values were shifted from -96 mV in the wild-type enzyme to -123 mV in D242S, -162 mV in D242E, -82 mV in F190L, -173 mV in F190V, and -51 mV in F190W. Collectively, these results demonstrate that D242 and F190 in MnP influence the electronic environment around the heme and that the reactions of compound II are far more sensitive to this influence than the reduction of compound I.     

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.     

Calculation of pK(a) in proteins with the microenvironment modulated-screened coulomb potential

The MM-SCP has been applied to predict pK(a) values of titratable residues in wild type and mutants of staphylococcal nuclease (SNase). The calculations were based on crystal structures made available by the Garcia-Moreno Laboratory. In the mutants, mostly deeply buried hydrophobic residues were replaced with ionizable residues, and thus their pK(a) values could be measured and calculated using various methods. The data set used here consisted of a set of WT SNase for which His pK(a) at several ionic strengths had been measured, a set of mutants for which measured pK(a) were available and a set of 11 mutants for which the measured pK(a) were not known at the time of calculation. For this latter set, blind predictions were submitted to the protein pK(a) cooperative, 2009 workshop at Telluride, where the results of the blind predictions were discussed (the RMSD of the submitted set was 1.10 pH units). The calculations on the structures with known pK(a) indicated that in addition to weaknesses of the method, structural issues were observed that led to larger errors (>1) in pK(a) predictions. For example, different crystallographic conditions or steric clashes can lead to differences in the local environment around the titratable residue, which can produce large differences in the calculated pK(a) . To gain further insight into the reliability of the MM-SCP, pK(a) of an extended set of 54 proteins belonging to several structural classes were carried out. Here some initial results from this study are reported to help place the SNase results in the appropriate context.     

Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4-11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.     

Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase

Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.     

Cytochromes: Reactivity of the "dark side" of the heme

Ligand binding to the heme distal side is a paradigm of heme-protein biochemistry, the proximal axial ligand being in most cases a His residue. NO binds to the ferrous heme-Fe-atom giving rise to hexa-coordinated adducts (as in myoglobin and hemoglobin) with His and NO as proximal and distal axial ligands, respectively, or to penta-coordinated adducts (as in soluble guanylate cyclase) with NO as the axial distal ligand. Recently, the ferrous derivative of Alcaligenes xylosoxidans cytochrome c' (Axcyt c') and of cardiolipin-bound horse heart cytochrome c (CL-hhcyt c) have been reported to bind NO to the "dark side" of the heme (i.e., as the proximal axial ligand) replacing the endogenous ligand His. Conversely, CL-free hhcyt c behaves as ferrous myoglobin by binding NO to the heme distal side, keeping His as the proximal axial ligand. Moreover, the ferrous derivative of CL-hhcyt c binds CO at the heme distal side, the proximal axial ligand being His. Furthermore, CL-hhcyt c shows peroxidase activity. In contrast, CL-free hhcyt c does not bind CO and does not show peroxidase activity. This suggests that heme-proteins may utilize both sides of the heme for ligand discrimination, which appears to be modulated allosterically. Here, structural and functional aspects of NO binding to ferrous Axcyt c' and (CL-)hhcyt c are reviewed.     

Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.     

New insights into the heme cavity structure of catalase-peroxidase: a spectroscopic approach to the recombinant synechocystis enzyme and selected distal cavity mutants

Catalase-peroxidases (KatGs) are heme peroxidases with homology to yeast cytochrome cperoxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs exhibit a peroxidase activity of broad specificity and a high catalase activity, which strongly depends on the presence of a distal Trp as part of the conserved amino acid triad Arg-Trp-His. By contrast, both CCP and APX do not have a substantial catalase activity despite the presence of the same triad. Thus, to elucidate structure-function relationships of catalase-peroxidases (for which no crystal structure is available at the moment), we performed UV-Vis and resonance Raman studies of recombinant wild-type KatG from the cyanobacterium SynechocystisPCC 6803 and the distal side variants (His123-->Gln, Glu; Arg119-->Ala, Asn; Trp122-->Phe, Ala). The distal cavity of KatG is very similar to that of the other class I peroxidases. A H-bond network involving water molecules and the distal Trp, Arg, and His is present, which connects the distal and proximal sides of the heme pocket. However, distal mutation not only affects the heme Fe coordination state and perturbs the proximal Fe-Im bond, as previously observed for other peroxidases, but also alters the stability of the heme architecture. The charge of the distal residues appears particularly important for maintaining the heme architecture. Moreover, the Trp plays a significant role in the distal H-bonding, much more pronounced than in CCP. The relevance of these findings for the catalase activity of KatG is discussed in light of the complete loss of catalase activity in the distal Trp mutants.     

Estimating the pKa values of basic and acidic side chains in ion channels using electrophysiological recordings: a robust approach to an elusive problem

As a step toward gaining a better understanding of the physicochemical bases of pK(a)-value shifts in ion channels, we have previously proposed a method for estimating the proton affinities of systematically engineered ionizable side chains from the kinetic analysis of single-channel current recordings. We reported that the open-channel current flowing through mutants of the (cation-selective) muscle nicotinic acetylcholine receptor (AChR) engineered to bear single basic residues in the transmembrane portion of the pore domain fluctuates between two levels of conductance. Our observations were consistent with the idea that these fluctuations track directly the alternate protonation-deprotonation of basic side chains: protonation of the introduced basic group would attenuate the single-channel conductance, whereas its deprotonation would restore the wild-type-like level. Thus, analysis of the kinetics of these transitions was interpreted to yield the pK(a) values of the substituted side chains. However, other mechanisms can be postulated that would also be consistent with some of our findings but according to which the kinetic analysis of the fluctuations would not yield true pK(a)s. Such mechanisms include the pH-dependent interconversion between two conformations of the channel that, while both ion permeable, would support different cation-conduction rates. In this article, we present experimental evidence for the notion that the fluctuations of the open-channel current observed for the muscle AChR result from the electrostatic interaction between fixed charges and the passing cations rather than from a change in conformation. Hence, we conclude that bona fide pK(a) values can be obtained from single-channel recordings.     

Energetics of proton transfer pathways in reaction centers from Rhodobacter sphaeroides. The Glu-H173 activated mutants

Electron transfer between the primary and secondary quinones (Q(A), Q(B)) in the bacterial photosynthetic reaction center (bRC) is coupled with proton uptake at Q(B). The protons are conducted from the cytoplasmic side, probably with the participation of two water channels. Mutations of titratable residues like Asp-L213 to Asn (inhibited mutant) or the double mutant Glu-L212 to Ala/Asp-L213 to Ala inhibit these electron transfer-coupled proton uptake events. The inhibition of the proton transfer (PT) process in the single mutant can be restored by a second mutation of Arg-M233 to Cys or Arg-H177 to His (revertant mutant). These revertant mutants shed light on the location of the main proton transfer pathway of wild type bRC. In contrast to the wild type and inhibited mutant bRC, the revertant mutant bRC showed notable proton uptake at Glu-H173 upon formation of the Q(B)- state. In all of these mutants, the pK(a) of Asp-M17 decreased by 1.4-2.4 units with respect to the wild type bRC, whereas a significant pK(a) upshift of up to 5.8 units was observed at Glu-H122, Asp-H170, Glu-H173, and Glu-H230 in the revertant mutants. These residues belonging to the main PT pathway are arranged along water channel P1 localized mainly in subunit H. bRC possesses subunit H, which has no counterpart in photosystem II. Thus, bRC may possess alternative PT pathways involving water channels in subunit H, which becomes active in case the main PT pathway is blocked.     

Transient spectroscopy of the reaction of cyanide with ferrous myoglobin. Effect of distal side residues

The reaction of cyanide metmyoglobin with dithionite conforms to a two-step sequential mechanism with formation of an unstable intermediate, identified as cyanide bound ferrous myoglobin. This reaction was investigated by stopped-flow time resolved spectroscopy using different myoglobins, i.e. those from horse heart, Aplysia limacina buccal muscle, and three recombinant derivatives of sperm whale skeletal muscle myoglobin (Mb) (the wild type and two mutants). The myoglobins from horse and sperm whale (wild type) have in the distal position (E7) a histidyl residue, which is missing in A. limacina Mb as well as the two sperm whale mutants (E7 His----Gly and E7 His----Val). All these proteins in the reduced form display an extremely low affinity for cyanide at pH less than 10. The differences in spectroscopy and kinetics of the ferrous cyanide complex of these myoglobins indicate a role of the distal pocket on the properties of the complex. The two mutants of sperm whale Mb are characterized by a rate constant for the decay of the unstable intermediate much faster than that of the wild type, at all pH values explored. Therefore, we envisage a specific role of the distal His (E7) in controlling the rate of cyanide dissociation and also find that this effect depends on the protonation of a single ionizable group, with pK = 7.2, attributed to the E7 imidazole ring. The results on A. limacina Mb, which displays the slowest rate of cyanide dissociation, suggests that a considerable stabilizing effect can be exerted by Arg E10 which, according to Bolognesi et al. (Bolognesi, M., Coda, A., Frigerio, F., Gatti, C., Ascenzi, P., and Brunori, M. (1990) J. Mol. Biol. 213, 621-625), interacts inside the pocket with fluoride bound to the ferric heme iron. A mechanism of control for the rate of dissociation of cyanide from ferrous myoglobin, involving protonation of the bound anion, is discussed.     

Zn(2+) binding properties of single-point mutants of the C-terminal zinc finger of the HIV-1 nucleocapsid protein: evidence of a critical role of cysteine 49 in Zn(2+) dissociation

The two highly conserved Zn(2+) finger motifs of the HIV-1 nucleocapsid protein, NCp7, strongly bind Zn(2+) through coordination of one His and three Cys residues. To further analyze the role of these residues, we investigated the Zn(2+) binding and acid-base properties of four single-point mutants of a short peptide corresponding to the distal finger motif of NCp7. In each mutant, one Zn(2+)-coordinating residue is substituted with a noncoordinating one. Using the spectroscopic properties of Co(2+), we first establish that the four mutants retain their ability to bind a metal cation through a four- or five-coordinate geometry with the vacant ligand position(s) presumably occupied by water molecule(s). Moreover, the pK(a) values of the three Cys residues of the mutant apopeptide where His44 is substituted with Ala are found by (1)H NMR to be similar to those of the native peptide, suggesting that the mutations do not affect the acid-base properties of the Zn(2+)-coordinating residues. The binding of Zn(2+) was monitored by using the fluorescence of Trp37 as an intrinsic probe. At pH 7.5, the apparent Zn(2+) binding constants (between 1.6 x 10(8) and 1.3 x 10(10) M(-)(1)) of the four mutants are strongly reduced compared to those of the native peptide but are similar to those of various host Zn(2+) binding proteins. As a consequence, the loss of viral infectivity following the mutation of one Zn(2+)-coordinating residue in vivo may not be related to the total loss of Zn(2+) binding. The pH dependence of Zn(2+) binding indicates that the coordinating residues bind Zn(2+) stepwise and that the free energy provided by the binding of a given residue may be modulated by the entropic contribution of the residues already bound to Zn(2+). Finally, the pK(a) of Cys49 in the holopeptide is found to be 5.0, a value that is at least 0.7 unit higher than those for the other Zn(2+)-coordinating residues. This implies that Cys49 may act as a switch for Zn(2+) dissociation in the distal finger motif of NCp7, a feature that may contribute to the high susceptibility of Cys49 to electrophilic attack.     

Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.     

Rearrangement of charge-charge interactions in variant ubiquitins as detected by double-mutant cycles and NMR

Previous studies of ubiquitin disclosed numerous charge-charge interactions on the protein's surface. To investigate how neighboring residues influence the strength of these interactions, double-mutant cycles are combined with pK(a) determinations by 2D NMR. More specifically, the environment around the Asp21-Lys29 ion pair has been altered through mutations at position 25, which is an asparagine in mammalian ubiquitin and a positively-charged residue in many other ubiquitin-like proteins. The pK(a) value of Asp21 decreases by 0.4 to 0.7 pH unit when Asn25 is substituted with a positively charged residue, suggesting a new and favorable ion pair interaction between positions 21 and 25. However, analysis of double mutants reveals that the favorable interaction between Asp21 and Lys29 is weakened when position 25 is a positively charged residue. Interestingly, while the pK(a) value of His25 in the N25H variant agrees with model compound values, additional mutants reveal that this agreement is fortuitous, resulting from a balance of favorable and unfavorable interactions; similar results were observed previously for Glu34 in ubiquitin and His8 in staphylococcal nuclease. Ionizable groups may thus have pK(a) values similar to model compound values and yet still be involved in significant interactions with other protein groups. One surprising result of introducing positively charged residues at position 25 is a new interaction between Lys29 and Glu18, an interaction not present in wild-type ubiquitin. This unanticipated result illustrates a key advantage of using NMR to determine pK(a) values for many residues simultaneously in the variant proteins. Overall, the strength of an interaction between two residues at the surface of ubiquitin is sensitive to the identity of neighboring residues. The results also demonstrate that relatively conservative and common point mutations such as substitutions of polar with charged residues and vice versa can have effects on interactions beyond the site of mutation per se.     

Roles of the heme proximal side residues tryptophan409 and tryptophan421 of neuronal nitric oxide synthase in the electron transfer reaction

Nitric oxide synthase (NOS) has an oxygenase domain with a thiol-coordinated heme active side similar to cytochrome P450. In contrast to cytochrome P450, however, conserved aromatic amino acids are situated in the heme proximal side of NOS. For example, in endothelial NOS (eNOS), the indole-ring nitrogen of Trp180 hydrogen-binds to the thiol of Cys186, the internal axial ligand to the heme. And, the aromatic side chain of Trp192 forms a bridge between this residue and the protein. Trp180 and Trp192 of eNOS correspond to Trp409 and Trp421 of neuronal NOS (nNOS), respectively. In order to understand the roles of the aromatic amino acids in catalysis, we generated Trp409His, Trp409Leu, Trp421His and Trp421Leu mutants of nNOS and determined their catalytic parameters. The Trp409Leu mutant was very poorly expressed in E. coli and was easily denatured during purification procedures. The NO formation activities of the Trp409His and Trp421Leu mutants were 11 and 25 micromol/min per micromol heme, respectively, and are lower than that (44 micromol/min per micromol heme) of the wild type. The activity (46 micromol/min per micromol heme) of the Trp421His mutant was comparable to that of the wild-type enzyme. However, NADPH oxidation rates of Trp421His (230 micromol/min per micromol heme) and Trp421Leu (104 micromol/min per microol heme) in the presence of L-Arg were much larger than those observed for the wild type (65 micromol/min per micromol heme) and the Trp409His mutant (43 micromol/min per micromol heme). The cytochrome c reduction rate of the Trp421His mutant was 6-fold larger than that of the wild type. The heme reduction rate with NADPH for the Trp421His mutant (0.09 min(-1)) was much lower than that (1.0 min(-1)) of the wild type. Taken together, it appears that Trp421 may be involved in inter-domain/inter-subunit electron transfer reactions.     

NMR determination of pKa values in α-synuclein

The intrinsically unfolded protein α-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK(a) values for all 26 sites in the protein that ionize below pH 7 using 2D (1) H-(15) N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK(a) values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK(a) values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For α-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK(a) of His50, a residue of particular interest since it is flanked within one turn of the α-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK(a) of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK(a) shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK(a) values of uncomplexed α-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding.     

pK values of histidine residues in ribonuclease Sa: effect of salt and net charge

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.     

Understanding how the distal environment directs reactivity in chlorite dismutase: spectroscopy and reactivity of Arg183 mutants

The chlorite dismutase from Dechloromonas aromatica (DaCld) catalyzes the highly efficient decomposition of chlorite to O(2) and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O-O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV-visible and resonance Raman spectroscopies showed that DaCld(R183A) contains five-coordinate high-spin (5cHS) heme, the DaCld(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and DaCld(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits pK(a) values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT DaCld the pK(a) of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid-base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis.     

pK(a) calculations for class C beta-lactamases: the role of Tyr-150

The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases. In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pK(a) values were also computed in catalytically impaired beta-lactamase mutants. Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation. In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed.     

Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments.