Probing electrostatic interactions along the reaction pathway of a glycoside hydrolase: histidine characterization by NMR spectroscopy

We have characterized by NMR spectroscopy the three active site (His80, His85, and His205) and two non-active site (His107 and His114) histidines in the 34 kDa catalytic domain of Cellulomonas fimi xylanase Cex in its apo, noncovalently aza-sugar-inhibited, and trapped glycosyl-enzyme intermediate states. Due to protection from hydrogen exchange, the level of which increased upon inhibition, the labile 1Hdelta1 and 1H epsilon1 atoms of four histidines (t1/2 approximately 0.1-300 s at 30 degrees C and pH approximately 7), as well as the nitrogen-bonded protons in the xylobio-imidazole and -isofagomine inhibitors, could be observed with chemical shifts between 10.2 and 17.6 ppm. The histidine pKa values and neutral tautomeric forms were determined from their pH-dependent 13C epsilon1-1H epsilon1 chemical shifts, combined with multiple-bond 1H delta2/epsilon1-15N delta1/epsilon2 scalar coupling patterns. Remarkably, these pKa values span more than 8 log units such that at the pH optimum of approximately 6 for Cex activity, His107 and His205 are positively charged (pKa > 10.4), His85 is neutral (pKa < 2.8), and both His80 (pKa = 7.9) and His114 (pKa = 8.1) are titrating between charged and neutral states. Furthermore, upon formation of the glycosyl-enzyme intermediate, the pKa value of His80 drops from 7.9 to <2.8, becoming neutral and accepting a hydrogen bond from an exocyclic oxygen of the bound sugar moiety. Changes in the pH-dependent activity of Cex due to mutation of His80 to an alanine confirm the importance of this interaction. The diverse ionization behaviors of the histidine residues are discussed in terms of their structural and functional roles in this model glycoside hydrolase.     

The H2 sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site

[NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H(2)-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H(2) sensor (RH) of Ralstonia eutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(epsilon) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.     

13C]Methylated ribonuclease A. 13C NMR studies of the interaction of lysine 41 with active site ligands

A unique resonance in the 13C NMR spectrum of [13C]methylated ribonuclease A has been assigned to a N epsilon, N-dimethylated active site residue, lysine 41. The chemical shift of this resonance was studied over the pH range 3 to 11, and the titration curve showed two inflection points, at pH 5.7 and 9.0. The higher pKa, designated pKa1, was assigned to the ionization of the lysyl residue itself while the pKa of 5.7, designated pKa2, was assigned on the basis of its pKa to the ionization of a histidyl residue which is somehow coupled to lysine 41. Both pKa values are measurably perturbed by the binding of active site ligands including nucleotides, nucleosides, phosphate, and sulfate. In most cases, the alterations in pKa values induced by the ligands were larger for pKa2. The ligand-induced perturbations in pKa2 generally paralleled those reported for histidine 12, another active site residue (Griffin, J. H., Schechter, A. N., and Cohen, J. S. (1973) Ann. N. Y. Acad. Sci. 222, 693-708). The sensitivity of the N epsilon, N-dimethylated lysine 41 resonance to the histidyl ionization may result from a conformational change in the active site region of ribonuclease which is coupled to the histidyl ionization. This coupling between lysine 41 and another ribonuclease residue, which has not been documented previously, offers new insight into the interrelationship between residues in the active site of this well characterized enzyme.     

Theoretical study of the discrimination between O(2) and CO by myoglobin

Combined quantum chemical and molecular mechanics geometry optimisations have been performed on myoglobin without or with O(2) or CO bound to the haem group. The results show that the distal histidine residue is protonated on the N(epsilon 2) atom and forms a hydrogen bond to the haem ligand both in the O(2) and the CO complexes. We have also re-refined the crystal structure of CO[bond]myoglobin by a combined quantum chemical and crystallographic refinement. Thereby, we probably obtain the most accurate available structure of the active site of this complex, showing a Fe[bond]C[bond]O angle of 171 degrees, and Fe[bond]C and C[bond]O bond lengths of 170-171 and 116-117 pm. The resulting structures have been used to calculate the strength of the hydrogen bond between the distal histidine residue and O(2) or CO in the protein. This amounts to 31-33 kJ/mol for O(2) and 2-3 kJ/mol for CO. The difference in hydrogen-bond strength is 21-22 kJ/mol when corrected for entropy effects. This is slightly larger than the observed discrimination between O(2) or CO by myoglobin, 17 kJ/mol. We have also estimated the strain of the active site inside the protein. It is 2-4 kJ/mol larger for the O(2) complex than for the CO complex, independent of which crystal structure the calculations are based on. Together, these results clearly show that myoglobin discriminates between O(2) and CO mainly by electrostatic interactions, rather than by steric strain.     

Solution structure of the low-molecular-weight protein tyrosine phosphatase from Tritrichomonas foetus reveals a flexible phosphate binding loop

Eukaryotic low-molecular-weight protein tyrosine phosphatases (LMW PTPs) contain a conserved serine, a histidine with an elevated pKa, and an active site asparagine that together form a highly conserved hydrogen bonding network. This network stabilizes the active site phosphate binding loop for optimal substrate binding and catalysis. In the phosphatase from the bovine parasite Tritrichomonas foetus (TPTP), both the conserved serine (S37) and asparagine (N14) are present, but the conserved histidine has been replaced by a glutamine residue (Q67). Site-directed mutagenesis, kinetic, and spectroscopic experiments suggest that Q67 is located near the active site and is important for optimal catalytic activity. Kinetic experiments also suggest that S37 participates in the active site/hydrogen bonding network. Nuclear magnetic resonance spectroscopy was used to determine the three-dimensional structure of the TPTP enzyme and to further examine the roles of S37 and Q67. The backbone conformation of the TPTP phosphate binding loop is nearly superimposable with that of other tyrosine phosphatases, with N14 existing in a strained, left-handed conformation that is a hallmark of the active site hydrogen bonding network in the LMW PTPs. As expected, both S37 and Q67 are located at the active site, but in the consensus structure they are not within hydrogen bonding distance of N14. The hydrogen bond interactions that are observed in X-ray structures of LMW PTPs may in fact be transient in solution. Protein dynamics within the active site hydrogen bonding network appear to be affected by the presence of substrate or bound inhibitors such as inorganic phosphate.     

15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism

Nitrogen-15 NMR spectroscopy has been used to study the hydrogen-bonding interactions involving the histidyl residue in the catalytic triad of alpha-lytic protease in the resting enzyme and in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The 15N shifts indicate that a strong hydrogen bond links the active site histidine and serine residues in the resting enzyme in solution. This result is at odds with interpretations of the X-ray diffraction data of alpha-lytic protease and of other serine proteases, which indicate that the serine and histidine residues are too far apart and not properly aligned for the formation of a hydrogen bond. In addition, the nitrogen-15 shifts demonstrate that protonation of the histidine imidazole ring at low pH in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate triggers the disruption of the aspartate-histidine hydrogen bond. These results suggest a catalytic mechanism involving directed movement of the imidazole ring of the active site histidyl residue.     

Neutral imidazole is the electrophile in the reaction catalyzed by triosephosphate isomerase: structural origins and catalytic implications

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, 13C and 15N NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. 15N NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pKa of histidine-95 is below 4.5. This abnormally low pKa rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. 15N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N epsilon 2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substitution of aspartate and glutamate for active center histidines in the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system maintain phosphotransfer potential

The active center histidines of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system proteins; histidine-containing protein, enzyme I, and enzyme IIA(Glc) were substituted with a series of amino acids (serine, threonine, tyrosine, cysteine, aspartate, and glutamate) with the potential to undergo phosphorylation. The mutants [H189E]enzyme I, [H15D]HPr, and [H90E]enzyme IIA(Glc) retained ability for phosphorylation as indicated by [(32)P]phosphoenolpyruvate labeling. As the active center histidines of both enzyme I and enzyme IIA(Glc) undergo phosphorylation of the N(epsilon2) atom, while HPr is phosphorylated at the N(delta1) atom, a pattern of successful substitution of glutamates for N(epsilon2) phosphorylations and aspartates for N(delta1) phosphorylations emerges. Furthermore, phosphotransfer between acyl residues: P-aspartyl to glutamyl and P-glutamyl to aspartyl was demonstrated with these mutant proteins and enzymes.     

His ... Asp catalytic dyad of ribonuclease A: histidine pKa values in the wild-type, D121N, and D121A enzymes

Bovine pancreatic ribonuclease A (RNase A) has a conserved His ... Asp catalytic dyad in its active site. Structural analyses had indicated that Asp121 forms a hydrogen bond with His119, which serves as an acid during catalysis of RNA cleavage. The enzyme contains three other histidine residues including His12, which is also in the active site. Here, 1H-NMR spectra of wild-type RNase A and the D121N and D121A variants were analyzed thoroughly as a function of pH. The effect of replacing Asp121 on the microscopic pKa values of the histidine residues is modest: none change by more than 0.2 units. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. In the presence of the reaction product, uridine 3'-phosphate (3'-UMP), protonation of one active-site histidine residue favors protonation of the other. This finding is consistent with the phosphoryl group of 3'-UMP interacting more strongly with the two active-site histidine residues when both are protonated. Comparison of the titration curves of the unliganded enzyme with that obtained in the presence of different concentrations of 3'-UMP shows that a second molecule of 3'-UMP can bind to the enzyme. Together, the data indicate that the aspartate residue in the His ... Asp catalytic dyad of RNase A has a measurable but modest effect on the ionization of the adjacent histidine residue.     

Tryptophan interactions in bacteriorhodopsin: a heteronuclear solid-state NMR study

The bulky and amphiphilic nature of tryptophan residues makes them particularly interesting components of proteins. In bacteriorhodopsin, four of the eight tryptophan residues are in the active site, forming parts of the retinal binding pocket. In this work, we use solid-state NMR to study the interactions of the tryptophan residues in wild-type bacteriorhodopsin, in the resting state, and in critical intermediates of the proton-motive photocycle. The range of the chemical shifts of the indole nitrogens suggests that all eight of them are hydrogen bonded. Using difference spectroscopy, we isolate several changes in these hydrogen bonds in the early and late M states. As found earlier for the peptide backbone, some perturbations found in the early M state relax in the transition to the late M state while new perturbations arise. Interestingly, Rotational Echo DOuble Resonance (REDOR) difference spectroscopy of [20-13C]retinal,[indole-15N]Trp-bR shows that indole of Trp182 is not involved in the significant hydrogen bond perturbations. We also use REDOR to measure dipolar interactions in [20-13C]retinal,[indole-15N]Trp-bR, and thereby determine the distance between the C20 of retinal and the indole nitrogen of Trp182. The internuclear distance changes only slightly from the light-adapted state (3.36 +/- 0.2 A) to the early M state (3.16 +/- 0.4 A).     

Ionization properties of titratable groups in ribonuclease T1. I. pKa values in the native state determined by two-dimensional heteronuclear NMR spectroscopy

pKa values of amino acid side chains of ribonuclease T1 have been determined from the pH dependence of 13C and 15N resonances. It was possible to derive pKa values of single protonation or deprotonation sites of carboxylate and imidazole groups. Deviations from pKa values of free amino acids could be interpreted with electrostatic interactions of corresponding side chains with the protein environment. In particular, the interaction between H27 and E82 led to an increase of the H27 pKa and a decrease of the E82 pKa. The pKa of E28 at the C-terminal end of the alpha-helix was increased because of the dipolar character of the alpha-helix. D76 did not titrate in the investigated pH range of about 2-9. From the chemical shift value this buried side chain seems to be protonated. The pKa values of side chains in the active site deviate from a normal behaviour. The lower pKa value of E58 may be interpreted with the close proximity of this side chain with positively charged H40 and R77. A novel two-dimensional 1H(13Cdelta)13Cgamma correlation experiment was developed to observe the pH dependence of the chemical shifts of the Cgamma resonances of histidine residues. From the inspection of the Cgamma chemical shift-pH profiles it was possible to determine the predominant tautomeric form for the histidine residues at higher pH values.     

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 1. Evidence from deuterium NMR measurements.

Deuterium NMR has been used to investigate the structure and dynamic state of cytochrome c complexed with bilayers of cardiolipin. Reductive methylation was employed to prepare [N epsilon, N epsilon-C2H3]lysyl cytochrome c, and deuterium exchange provided labeling of backbone sites to give [amide-2H]cytochrome c or more selective labeling of just histidine residues in [epsilon-2H]histidine cytochrome c. Deuterium NMR measurements on [N epsilon, N epsilon-C2H3]lysyl cytochrome c in the solid state showed restricted motions, fairly typical of the behavior of aliphatic side-chain sites in proteins. The [amide-2H]cytochrome c provided "immobile" amide spectra showing that only the most stable backbone sites remained labeled in this derivative. Relaxation measurements on the aqueous solution of [amide-2H]cytochrome c yielded a rotational correlation time of 7.9 ns for the protein, equivalent to a hydrodynamic diameter of 4.0 nm, just 0.6 nm greater than its largest crystallographic dimension. Similar measurements on [epsilon-2H]histidine cytochrome c in solution showed that all labeled histidine residues were also "immobile" compared with the overall reorientational motion of the protein. The interaction with cardiolipin bilayers appeared to create a high degree of mobility for the side-chain sites of [N epsilon, N epsilon-C2H3]lysyl cytochrome c and perturbed backbone structure to instantaneously release all deuterons in [amide-2H]cytochrome c. The [epsilon-2H]histidine cytochrome c derivative, when complexed with cardiolipin, failed to produce any detectable wide-line 2H NMR spectrum, demonstrating that the overall reorientational motion of bound protein was not isotropic on the NMR time scale, i.e., tau c greater than 10(-7)s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315.

The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H.     

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.     

Refined X-ray structure of the low pH form of ribonuclease T1-2'-guanylic acid complex at 1.9 A resolution

The three-dimensional X-ray structure of the RNase T1[EC 3.1.27.3]-2'GMP complex crystallized at low pH value (4.0) was determined, and refined to 1.9 A resolution to give a final R value of 0.203. The refined model includes 781 protein atoms, 24 inhibitor atoms, and 43 solvent molecules. The imidazole rings of His27 and His40 interact with the carboxyl side chains of Glu82 and Glu58, respectively, whereas that of His92 is in contact with the main chain carbonyl oxygen of Ala75. In the complex, the ribose ring of the 2'GMP molecule adopts a C2'-endo puckering, and the exocyclic conformation is gauche(-)-gauche(+). The glycosyl torsion angle is in the syn range with an intramolecular hydrogen bond between N3 and O5', and the 2'-phosphate orientation is trans-gauche(-). The guanine base of the inhibitor is tightly bound to the base recognition site with five hydrogen bonds (N1--Glu46O epsilon 2, N2---Asn98O,O6---Asn44N, and N7 ---Asn43N delta 2/Asn43N) and is sandwiched between the phenolic ring portions of Tyr42 and Tyr45 by stacking interactions. The 2'-phosphate group interacts with Arg77N eta 2, Glu58O episilon 2, and Tyr 38O eta but not with any of the histidine residues. Arg77N eta 2 also interacts with Tyr38O eta. There is no interaction between the ribose moiety of the inhibitor and the enzyme.     

Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond.

The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C11-P-Y-C14-H-. The 1H NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys-11 thiolate, which is shown to have an abnormally low pKa value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-11 thiolate.     

Tautomerism,acid-base equilibria,and H-bonding of the six histidines in subtilisin BPN' by NMR

We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.     

Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.     

The fe2+ site of photosynthetic reaction centers probed by multiple scattering x-ray absorption fine structure spectroscopy: improving structure resolution in dry matrices

We report on the x-ray absorption fine structure of the Fe(2+) site in photosynthetic reaction centers from Rhodobacter sphaeroides. Crystallographic studies show that Fe(2+) is ligated with four N(epsilon) atoms from four histidine (His) residues and two O(epsilon) atoms from a Glu residue. By considering multiple scattering contributions to the x-ray absorption fine structure function, we improved the structural resolution of the site: His residues were split into two groups, characterized by different Fe-N(epsilon) distances, and two distinct Fe-O(epsilon) bond lengths resolved. The effect of the environment was studied by embedding the reaction centers into a polyvinyl alcohol film and into a dehydrated trehalose matrix. Incorporation into trehalose caused elongation in one of the two Fe-N(epsilon) distances, and in one Fe-O(epsilon) bond length, compared with the polyvinyl alcohol film. The asymmetry detected in the cluster of His residues and its response to incorporation into trehalose are ascribed to the hydrogen bonds between two His residues and the quinone acceptors. The structural distortions observed in the trehalose matrix indicate a strong interaction between the reaction-centers surface and the water-trehalose matrix, which propagates deeply into the interior of the protein. The absence of matrix effects on the Debye-Waller factors is brought back to the static heterogeneity and rigidity of the ligand cluster.     

Quantitative identification of the protonation state of histidines in vitro and in vivo

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.