Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.     

Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen

A stable nonreducible trifunctional cross-linking amino acid has been isolated from mature bovine skin collagen fibrils. Previous cross-link peptide isolations and amino acid analyses indicate the compound has properties identical with those of hydroxyaldolhistidine. Its newly proposed structure was verified using fast atom bombardment mass spectrometry, and 1H and 13C nuclear magnetic resonance. The data indicated that the cross-link consists of the prosthetic groups from one residue each of histidine, hydroxylysine, and lysine. The 1H and 13C nuclear magnetic resonance data indicated that imidazole C-2 of histidine is linked to C-6 of norleucine (epsilon-deaminated lysine residue) which in turn is linked to the C-6 amino group of hydroxylysine. Based on the trivial names for other cross-linking residues found in collagen and elastin it was given the name histidinohydroxylysinonorleucine. In vitro incubation studies for up to 24 weeks, in aqueous solution at physiological pH and ionic strength, using 6-month-old bovine embryo skin demonstrated a one-to-one stoichiometric relationship between the disappearance of the labile reducible bifunctional cross-link dehydrohydroxylysinonorleucine and the appearance of histidinohydroxylsinonorleucine. These results can partially explain the previously observed disappearance of dehydrohydroxylysinonorleucine with chronological age.     

The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin

In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.     

Nuclear magnetic resonance titration curves of histidine ring protons. Conformational transition affecting three of the histidine residues of ribonuclease.

NMR titration curves are reported for the 4 histidine residues of ribonuclease A in sodium acetate and for ribonuclease S in sodium acetate, phosphate, and sulfate solutions. Evidence is presented that the imidazole side chain of histidine residue 48 undergoes a conformational change, probably also involving the carboxyl side chain of aspartic acid residue 14. This group is considered to be responsible for the low pH inflection with pKa 4.2 present in the NMR titration curve of the C-2 proton resonance of histidine 48. The NMR titration curves of the active site histidine residues 12 and 119 also exhibit inflections at low pH values, although there is no carboxyl group within 9 A of the imidazole side chain of histidine residue 12 in the structure of ribonuclease S determined by x-ray crystallography (Wyckoff, H. W., Tsernoglou, D., Hanson, A. W. Knox, J. R., Lee, B., and Richards, F. M. (1970) J. Biol. Chem. 245, 305-328). Curve fitting was carried out on 11 sets of NMR titration data using a model in which the 3 histidine residues 12, 119, and 48 are assumed to be affected by a common carboxyl group. The results obtained indicate that such a model with fewer parameters gives as good a representation of the data as the model in which each histidine residue is assumed to interact separately with a different carboxyl group. Therefore, it is concluded that the ionization of aspartic acid residue 14 is indirectly experienced by the active site histidine residues through the conformational change at histidine 48. A model assuming mutual interaction of the active site histidine residues does not account for the low pH inflections in these curves.     

Assignment of the histidine proton magnetic resonance peaks of soybean trypsin inhibitor (Kunitz) by a differertial deuterium exchange technique.

Deuterium exchange at the C(2)-H position of the two histidine residues of native soybean trypsin inhibitor (Kunitz) in 2-H2O was followed by 1-H nuclear magnetic resonance (NMR) spectroscopy. The two histidine residues of soybean trypsin inhibitor exchange at significantly different rates at pH* 5.00, 40 degrees. Half-times observed were: peak H1, t1/2=61 plus or minus 2 days; peak H2, T1/2=24 plus or minus 2 days. Differentially deuterated soybean trypsin inhibitor was cleaved by cyanogen bromide into two fragments each containing one histidine residue. The deuterium content of the histidine residue of each separated fragment was analyzed by 1H NMR spectroscopy. Hisidine-71 in fragment 1-114 showed approximately twice the deuterium content of His-157 in fragment 115-181. These results lead to the assignment of 1H NMR peak H1 to His-157 and peak H2 to His-71. These assignments were extended to the histidine peaks of trypsin-modified soybean trypsin inhibitor by converting the differentially deuterated virgin soybean trypsin inhibitor to the modified form. The correlation of histidine peaks in virgin amd modified soybean trypsin inhibitors was the same as proposed earlier on the basis of pK arguments. The results demonstrate that His-71 is the residue whose pK value is raised from 5.27 to 5.91 on trypsin modification of soybean trypsin inhibitor [Markley, J. L., (1973), Biochemistry 12, 2245].     

A proton-nuclear-magnetic-resonance study of human somatotropin (growth hormone). Assignment and properties of the histidine residues.

The 1H-n.m.r. spectra of human somatotropin (growth hormone) show perturbed peaks from individual aromatic and aliphatic apolar residues, characteristic of a specifically folded globular structure. The imidazole C-2-H resonances of the histidine residues (at positions 18, 21 and 151 in the somatotropin sequence) were individually resolved, and their titration behaviour in the pH range 1.2-11.5 was investigated. The imidazole C-2-H resonance of histidine-151 is assigned, by comparison of its titration behaviour in human somatotropin and desamido-somatotropin (Asn-152 leads to Asp-152). The C-2-H resonances of all three histidine residues are assigned, by comparison of their relative deuterium-exchange rates (determined by n.m.r.) and the relative tritium-exchange rates of the histidine residues (determined by tryptic digestion of tritiated human somatotropin and reversed-phase high-pressure liquid-chromatographic separation of the histidine-containing tryptic peptides). There is evidence that histidine-18 forms an ion-pair bond with a glutamic acid or aspartic acid residue. The globular structure does not appear to change from pH3 to 11.5, though there is evidence for an unfolding of a region of the structure (involving histidine-21 and a tyrosine residue) below pH3.     

Proton nuclear magnetic resonance studies of histidines in horse carbonic anhydrase I

The 250 MHz 1H-NMR spectrum of horse carbonic anhydrase I (or B) (carbonate hydro-lyase, EC 4.2.1.1) was measured as a function of pH under various conditions. Eight resonances corresponding to histidine C-2 protons and four resonances corresponding to histidine C-4 protons were identified and assigned to individual histidine residues in the enzyme molecule. Substantial similarities between horse and human carbonic anhydrases I were demonstrated. While the human enzyme has three titratable histidine residues in its active site, the horse enzyme has only two, His-67 in the human enzyme being replaced by Gln in the horse enzyme (Jabusch, J.R., Bray, R.P. and Deutsch, H.F. (1980) J. Biol. Chem. 255, 9196-9204). This substitution has small but significant effects on the behaviour of the other active-site histidines. His-64 and His-200. However, His-64 has an anomalously low pKa value also in horse isoenzyme I, as previously observed in human isoenzyme I (Campbell, I.D., Lindskog, S. and White, A.I. (1974) J. Mol. Biol. 90, 469-489).     

Guanyl-specific ribonuclease from the fungus Penicillium chrysogenum strain 152 and its complex with guanosine 3'-phosphate studied by nuclear magnetic resonance

The method of proton magnetic resonance was used to obtain information on the active site of the guanyl-specific ribonuclease from Penicillium chrysogenum, strain 152A. Four pH-dependent signals in the aromatic region of the proton NMR spectrum of the enzyme were assigned to the C-2 and C-4 protons of the two histidine residues. To determine the pK values and the environment of the histidine residues the pH dependence of their chemical shifts was studied and experimental curves thus obtained were analyzed taking into account the effect of other dissociating groups of the enzyme. The pK values of the histidine residues were found to be equal to 7.92 +/- 0.04 and 7.86 +/- 0.09. The results of the calculations indicate that each histidine residue should interact with an acidic group (carboxylic) of the protein (pK 4.33 and 3.48) and the distance between two histidine residues does not exceed 0.85 nm. The rate constants for the quasi-first order reaction of deuterium exchange of the histidine residues (11.2 s-1 and 3.7 x-1) suggest that both residues are accessible, though to a different degree to solvent. Formation of a complex between the enzyme and guanosine 3'-phosphate (Guo3'P) is accompanied by the shift of the histidine pK toward the alkaline region by 0.5. The existence of the complex is controlled by dissociation of a histidine residue with pK 8.7 in alkaline medium and by protonation of the N-7 of Guo3'P (pK 2.4) in acid medium. Nuclear Overhauser effect measurements were used to determine the glycosidic torsion angle for the Guo3'P in the complex and to estimate the distances between the histidine residues of the enzyme and ribose ring of Guo-3'P. The results obtained suggest that the nucleotide in the complex has an anti conformation and the least exposed histidine is spaced not more than 0.5 nm from the C-1' proton of the nucleotide ribose ring. A model for the enzyme-nucleotide complex is presented.     

Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A.

1H NMR spectroscopy at 100 MHz was used to determine the first-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35 degrees C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35 degrees C and pH meter reading 11 caused irreversible denaturation of RN-ase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem, (Tokyo) 75, 1197-1200 (1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5 The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936-937]. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26 degrees C. His-48 in S-protein titrates normally and has a lower pK than in RN-ase-A probably because of the absence of Asp-14, which in RN-ase-A forms a a hydrogen bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9.     

A retro-evolution study of CDP-6-deoxy-D-glycero-L-threo-4-hexulose-3-dehydrase (E1) from Yersinia pseudotuberculosis: implications for C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses

CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E1), which catalyzes C-3 deoxygenation of CDP-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other B6-dependent enzymes, albeit with two important distinctions. It is a rare example of a B6-dependent enzyme that harbors a [2Fe-2S] cluster, and a highly conserved lysine that serves as an anchor for PLP in most B6-dependent enzymes is replaced by histidine at position 220 in E1. Since alteration of His220 to a lysine residue may produce a putative progenitor of E1, the H220K mutant was constructed and tested for the ability to process the predicted substrate, CDP-4-amino-4,6-dideoxyglucose, using PLP as the coenzyme. Our data showed that H220K-E1 has no dehydrase activity, but can act as a PLP-dependent transaminase. However, the reaction is not catalytic since PLP cannot be regenerated during turnover. Reported herein are the results of this investigation and the implications for the role of His220 in the catalytic mechanism of E1.     

Selective abstraction of 2H from C-1' of the C residue in AGC.ICT by the radical center at C-2 of activated neocarzinostatin chromophore: structure of the drug/DNA complex responsible for bistranded lesion formation.

Glutathione-activated neocarzinostatin chromophore (NCS-Chrom) generates bistranded lesions at AGC.GCT sequences in DNA, consisting of an abasic site at the C residue and a strand break at the T residue on the complementary strand, due to hydrogen atom abstraction from C-1' and C-5', respectively. Earlier work showed that 2H from C-5' of T was selectively abstracted by the radical center at C-6 of activated NCS-Chrom, supporting a proposed model of the active-drug/DNA complex. However, since under the conditions used breaks at the T exceeded their inclusion in bistranded lesions, it was not clear what fraction of the hydrogen transfer represented bistranded lesions. Since virtually all abasic sites at the C are part of a bistranded lesions, hydrogen transfer from C-1' of C into the drug should reflect only the bistranded reaction. Accordingly, a self-complementary oligodeoxynucleotide 5'-GCAGCICTGC-3' was synthesized in which the C contained 2H at the C-1' position. In order to eliminate an 2H isotope effect on the transfer and to increase the extent of the bistranded reaction, an I residue was substituted for the G opposite the C residue. Sequencing gel electrophoretic analysis revealed that under one-hit kinetics, 37% of the damage reaction was associated with abasic site (alkali-labile break) formation at the C residue and 48% with direct strand breaks at the T residue. Thus, 74% of the damage involved a bistranded lesion. 1H NMR spectroscopic analysis of the reacted chromophore showed that 2H had been selectively transferred into the C-2 position to the extent of approximately 22%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of the histidine residues in the pH-induced conformational change of histone H5

Comparative studies on the conformational stability of histones H1 and H5 have been carried out by monitoring the pH-induced conformational transitions of the proteins by CD and 1H NMR spectroscopies. The transition point of H1 agrees with the pKa of the carboxyl groups of the acidic residues. In contrast, the transition of H5 is associated with the ionization of the histidine residues which exist exclusively in H5, as well as the deionization of the acidic residues. These observations, combined with the result of the deuterium exchange rates of the histidine C-2 protons, led us to conclude that His-25 and His-62, which are buried in the globular domain, play an important role in the conformational stability of histone H5.     

The 1H nuclear-magnetic-resonance spectroscopy of cobalt(II)-beta-lactamase II.

The 1H n.m.r. spectra of beta-lactamase II in the presence of Co(II) were studied. Analysis of the spectra suggests that Co(II) binds at the same two metal-binding sites as does Zn(II). The binding of Co(II) at the first site is much weaker than the binding of Zn(II) at this site, whereas the binding of Co(II) at the second site is tighter than the binding of Zn(II). The binding of Co(II) to the mono-zinc(II)-enzyme caused only one marked change in the spectrum, namely a decrease in the intensity of the resonances assigned to the C-2 and C-4 protons of one histidine residue (residue E). However, when the spectra of the apoenzyme and the Co(II)-enzyme were compared, there were many differences. A significant fraction of the protons in the whole molecule are affected by the binding of Co(II) at the first metal-ion-binding site (where the ligands are the enzyme's sole thiol group and three histidine residues). This may be because the first site is internal, or because of a difference in conformation between the apoenzyme and the mono-Co(II)-enzyme. The second site may be located on the surface of the molecule.     

The pentaammineruthenium(III)histidine complex in ribonuclease A: application to the assignment of histidine proton resonances

The assignment of two histidine proton resonances in the proton NMR spectrum of ribonuclease A has been made by forming a paramagnetic complex between pentaammineruthenium(III) and the N-3 nitrogen of a single histidine residue. Reaction of chloropentaammineruthenium(III)dichloride with ribonuclease A in 0.1 m Tris-HCl, pH 7.0, 25°C yields a variety of products in which various histidine residues have been labeled. Cation-exchange chromatography affords the isolation of a specific derivative, labeled at a single histidine residue, that retains 66% of the activity toward the hydrolysis of 2′,3′-cyclic CMP. The site of labeling was determined by peptide mapping to be histidine 105. The binding of ruthenium results in the disappearance of both a histidine C-2 and a C-4 proton resonance from the downfield region of the proton NMR spectrum, as expected from model compound studies. The assignment of these two resonances to histidine 105 is in agreement with a previous assignment (J. L. Markley, 1975, Biochemistry, 14, 3546–3554), thereby demonstrating the potential utility of this ruthenium reagent in the assignment of histidine resonances in the proton NMR spectra of other proteins.     

Proton NMR studies of the biologically active 1-34 fragment of bovine parathyroid hormone: examination of a structural model

Proton NMR spectra of the biologically active 1-34 fragment of bovine parathyroid hormone (bPTH) were studied as a function of pH over the range of pH 4 to 10, in buffer and in 6 M guanidine DC1. One of the histidine C-2 peaks titrated normally, with a pKa value of 6.8, but the other two histidines in this peptide had pKa values of 6.3. Denatured PTH showed only one histidine C-2 peak with a pKa of 6.7. An aliphatic peak identified as due to either a methionine or a glutamine residue also shifted with pH, and the pKa for this shift was 6.3. Finally, small but significant upfield shifts in the methyl and methylene resonances were observed as a function of pH, and when compared to the denatured peptide. These results indicate that the N-terminal domain of native PTH has considerable structure in solution, and are consistent with a theoretical model for the folding of this peptide.     

Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.     

Modifications of the pyroglutamic acid and histidine residues in thyrotropin-releasing hormone (TRH) yield analogs with selectivity for TRH receptor type 2 over type 1

Thyrotropin-releasing hormone (TRH) analogs in which the N-1(tau) or the C-2 position of the imidazole ring of the histidine residue is substituted with various alkyl groups and the l-pyroglutamic acid (pGlu) is replaced with the l-pyro-2-aminoadipic acid (pAad) or (R)- and (S)-3-oxocyclopentane-1-carboxylic acid (Ocp) were synthesized and studied as agonists for TRH receptor subtype 1 (TRH-R1) and subtype 2 (TRH-R2). We observed that several analogs were selective agonists of TRH-R2 showing relatively less or no activation of TRH-R1. For example, the most selective agonist of the series 13, in which pGlu is replaced with the pAad and histidine residue is substituted at the N-1 position with an isopropyl group, was found to activate TRH-R2 with a potency (EC(50)=1.9microM) but did not activate TRH-R1 (potency>100 microM); that is, exhibited >51-fold greater selectivity for TRH-R2 versus TRH-R1. Analog 8, in which pGlu is replaced with pAad and histidine is substituted at the N-1(tau) position with a methyl group, exhibited a binding affinity (K(i)=0.0032 microM) to TRH-R1 that is similar to that of [Ntau(1)-Me-His]-TRH and displayed potent activation of TRH-R1 and TRH-R2 (EC(50)=0.0049 and 0.0024 microM, respectively). None of the analogs in which pGlu is replaced with the bioisosteric (R)- and (S)-(Ocp) and the imidazole ring is substituted at the N-1(tau) or C-2 position were found to bind or activate either TRH-R1 or TRH-R2 at the highest test dose of 100 microM.     

Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.

In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H(2)O(2) or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically (13)C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H(2)O(2) solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.     

Identification of histidine residues that act as zinc ligands in beta-lactamase II by differential tritium exchange.

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.     

Electron paramagnetic resonance characterization of the copper-resistance protein PcoC from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Continuous-wave and pulsed electron paramagnetic resonance have been applied to the study of the Cu(II) site of the copper-resistance protein PcoC from Escherichia coli and certain variant forms. Electron spin echo envelope modulation (ESEEM) experiments confirm the presence of two histidine ligands, His1 and His92, at the Cu(II) site of wild-type PcoC, consistent with the available X-ray crystallographic data for the homolog CopC (67% sequence identity) from Pseudomonas syringae pv. tomato. The variants H1F and H92F each lack one of the histidine residues close to the Cu(II) site. The ESEEM data suggest that the surviving histidine residue remains as a ligand. The nA variant features an extra alanine residue at the N terminus, which demotes the His1 ligand to position 2. At least one of the two histidine residues is bound at the Cu(II) site in this form. Simulation of the (14)N superhyperfine structure in the continuous-wave spectra confirms the presence of at least three nitrogen-based ligands at the Cu(II) sites of the wild-type, H92F and nA forms, while the H1F variant has two nitrogen ligands. The spectra of wild-type form can be fitted adequately with a 3N or a 4N model. The former is consistent with the crystal structure of the CopC homolog, where His1 acts as a bidentate ligand. The latter raises the possibility of an additional unidentified nitrogen ligand. The markedly different spectra of the H1F and nA forms compared with the wild-type and H92F proteins further highlight the integral role of the N-terminal histidine residue in the high-affinity Cu(II) site of PcoC.