Protonation State Of Histidine Research Articles

Noncovalent Interactions Involving Histidine: The Effect of Charge on π−π Stacking and T-Shaped Interactions with the DNA Nucleobases

Detailed (gas-phase) MP2/6-31G*(0.25) potential energy surface scans and CCSD(T) energy calculations at the complete basis set (CBS) limit were used to analyze the (face-to-face) stacking and (edge-to-face) T-shaped interactions between histidine (modeled as imidazole) and the DNA nucleobases. For the first time, a variety of relative monomer arrangements between both neutral and protonated histidine and the natural nucleobases were considered to determine the effects of charge on the optimum dimer geometry and binding strength. Our results reveal that protonation of histidine changes the preferred relative orientations of the monomers and propose that these geometric differences may be combined with experimental crystal structures to assess the protonation state of histidine in different environments. It is also found that protonation affects the nucleobase binding preference, as well as the magnitude of the stacking and T-shaped interactions. Indeed, the maximum possible stacking and T-shaped interactions involving the neutral histidine range between approximately 20 and 45 kJ mol(-1), while this range increases to 40-105 kJ mol(-1) upon protonation, which represents an up to 330% enhancement. Although an increase in the interaction energies upon protonation of histidine is expected, the present work provides a measure of the magnitude of this enhancement in the gas phase and reveals that the amplification is almost entirely due to larger electrostatic contributions. The relative strengthening of different classifications of dimers upon protonation leads to stronger T-shaped interactions than stacking energies for protonated histidine, while the stacking and T-shaped interactions involving neutral histidine are of comparable magnitude. Thus, there is a significant difference in the nature of the pi(cation)-pi interactions involving protonated histidine and the pi-pi interactions involving neutral histidine. The calculated strengths of the interactions studied in the present work suggest that both neutral and cationic histidine contacts will provide significant stabilization to DNA-protein complexes. Although solvation effects will decrease the magnitude of the reported interactions, our results are applicable to a variety of low-polarity, biologically-relevant environments such as nonpolar enzyme active sites. Therefore, our calculations suggest that these interactions may also be important for many biological processes. The proposed significance of these interactions is supported by the large number of histidine-nucleobase contacts that appear in experimental crystal structures. The highly accurate (MP2/6-31G*(0.25)) preferred structures and (CCSD(T)/CBS) binding strengths reported in the present work can be used as benchmarks to analyze the performance of existing, or to develop new, molecular mechanics force fields for use in large-scale molecular dynamics (MD) studies of DNA-protein complexes.

Study of the Specific Lipid Binding Properties of Aβ 11−22 Fragment at Endosomal pH

Increasing evidence implicates interactions between Abeta peptide and lipids in the development of Alzheimer's disease. More generally, Abeta peptide interactions with membranes seem to depend on the composition of the lipid bilayer and the structural features of the peptide. One key parameter should be pH, since one site of intracellular Abeta peptide production and/or accumulation is likely to be endosomes. This intracellular endosomal accumulation was suggested to contribute to disease progression. In this paper, we report a study on the 11-22 amphiphilic domain of Abeta in interaction with model membrane; this region contains most of the charged residues of the N-terminal domain of Abeta. We show that the peptide charge, and more precisely the protonation state of histidines 13 and/or 14, is important for the interaction with lipids. Hence, it is only at endosomal pH that a conformational change of the peptide is observed in the presence of negatively charged lipid vesicles, that is, when both lipid headgroups and histidines can interact through electrostatic interactions. Specific interactions of the fragment with phosphatidylserine and to a lesser extent with phosphatidylcholine, but not phosphatidylethanolamine, are further evidenced by the Langmuir monolayer technique. From our results, we suggest that the protonation state of His residues could have a role in the pathogenic surface interaction of the whole Abeta peptide with membranes.

Ionization of Aqueous Cations: Photoelectron Spectroscopy and ab Initio Calculations of Protonated Imidazole

Photoelectron spectroscopy and ab initio calculations employing a nonequilibrium polarizable continuum model were employed for determining the vertical ionization potential of aqueous protonated imidazole. The experimental value of 8.96 eV is in in excellent agreement with calculations, which also perform quantitatively for ionization of aqueous alkali cations as benchmark species. The present results show that protonation of imidazole increases its vertical ionization potential up in water by 0.7 eV, which is significantly larger than the resolution of the experiment or the error of the calculation. This combined experimental and computational approach may open the possibility for quantitatively analyzing the protonation state of histidine, of which imidazole is the titratable side chain group, in aqueous peptides and proteins.

Unveiling the Role of Histidine and Tyrosine Residues on the Conformation of the Avian Prion Hexarepeat Domain

The prion protein (PrPC) is a glycoprotein that in mammals, differently from avians, can lead to prion diseases, by misfolding into a beta-sheet-rich pathogenic isoform (PrPSc). Mammal and avian proteins show different N-terminal tandem repeats: PHGGGWGQ and PHNPGY, both containing histidine, whereas tyrosine is included only in the primary sequence of the avian protein. Here, by means of potentiometric, circular dichroism (CD), and molecular dynamics (MD) studies at different pH values, we have investigated the conformation of the avian tetrahexarepeat (PHNPGY)4 (TetraHexaPY) with both N- and C-termini blocked by acetylation and amidation, respectively. We have found, also with the help of a recently proposed protein chirality indicator (Pietropaolo, A.; Muccioli, L.; Berardi, R.; Zannoni, C. Proteins 2008, 70, 667-677), a conformational dependence on the protonation states of histidine and tyrosine residues: the turn formation is pH driven, and at physiological pH a pivotal role is played by the tyrosine OH groups which give rise to a very compact bent structure of backbone upon forming a hydrogen-bond network.

The determination of protonation states in proteins

The protonation states of aspartic acids and glutamic acids as well as histidine are investigated in four X-ray cases: Ni,Ca concanavalin A at 0.94 A, a thrombin-hirugen binary complex at 1.26 A resolution and two thrombin-hirugen-inhibitor ternary complexes at 1.32 and 1.39 A resolution. The truncation of the Ni,Ca concanavalin A data at various test resolutions between 0.94 and 1.50 A provided a test comparator for the ;unknown' thrombin-hirugen carboxylate bond lengths. The protonation states of aspartic acids and glutamic acids can be determined (on the basis of convincing evidence) even to the modest resolution of 1.20 A as exemplified by our X-ray crystal structure refinements of Ni and Mn concanavalin A and also as indicated in the 1.26 A structure of thrombin, both of which are reported here. The protonation-state indication of an Asp or a Glu is valid provided that the following criteria are met (in order of importance). (i) The acidic residue must have a single occupancy. (ii) Anisotropic refinement at a minimum diffraction resolution of 1.20 A (X-ray data-to-parameter ratio of approximately 3.5:1) is required. (iii) Both of the bond lengths must agree with the expectation (i.e. dictionary values), thus allowing some relaxation of the bond-distance standard uncertainties required to approximately 0.025 A for a '3sigma' determination or approximately 0.04 A for a '2sigma' determination, although some variation of the expected bond-distance values must be allowed according to the microenvironment of the hydrogen of interest. (iv) Although the F(o) - F(c) map peaks are most likely to be unreliable at the resolution range around 1.20 A, if admitted as evidence the peak at the hydrogen position must be greater than or equal to 2.5 sigma and in the correct geometry. (v) The atomic B factors need to be less than 10 A(2) for bond-length differentiation; furthermore, the C=O bond can also be expected to be observed with continuous 2F(o) - F(c) electron density and the C-OH bond with discontinuous electron density provided that the atomic B factors are less than approximately 20 A(2) and the contour level is increased. The final decisive option is to carry out more than one experiment, e.g. multiple X-ray crystallography experiments and ideally neutron crystallography. The complementary technique of neutron protein crystallography has provided evidence of the protonation states of histidine and acidic residues in concanavalin A and also the correct orientations of asparagine and glutamine side chains. Again, the truncation of the neutron data at various test resolutions between 2.5 and 3.0 A, even 3.25 and 3.75 A resolution, examines the limits of the neutron probe. These various studies indicate a widening of the scope of both X-ray and neutron probes in certain circumstances to elucidate the protonation states in proteins.

Molecular Dynamics Simulations Show That Bound Mg2+ Contributes to Amino Acid and Aminoacyl Adenylate Binding Specificity in Aspartyl-tRNA Synthetase through Long Range Electrostatic Interactions

Molecular recognition between the aminoacyl-tRNA synthetase enzymes and their cognate amino acid ligands is essential for the faithful translation of the genetic code. In aspartyl-tRNA synthetase (AspRS), the co-substrate ATP binds preferentially with three associated Mg2+ cations in an unusual, bent geometry. The Mg2+ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP x Mg3(2+) complex was shown recently to increase the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted. Here, we used molecular dynamics free energy simulations and continuum electrostatic calculations to resolve two related questions. First, we showed that if one of the Mg2+ cations is removed, the Asp/Asn binding specificity is strongly reduced. Second, we computed the relative stabilities of the three-cation complex and the 2-cation complexes. We found that the 3-cation complex is overwhelmingly favored at ordinary magnesium concentrations, so that the protein is protected against the 2-cation state. In the homologous LysRS, the 3-cation complex was also strongly favored, but the third cation did not affect Lys binding. In tRNA-bound AspRS, the single remaining Mg2+ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg2+ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre- and post-adenylation states.

Assessing the acid–base and conformational properties of histidine residues in human prion protein (125–228) by means of pKa calculations and molecular dynamics simulations

A thorough study of the acid-base behavior of the four histidines and the other titratable residues of the structured domain of human prion protein (125-228) is presented. By using multi-tautomer electrostatic calculations, average titration curves have been built for all titratable residues, using the whole bundles of NMR structures determined at pH 4.5 and 7.0. According to our results, (1) only histidine residues are likely to be involved in the first steps of the pH-driven conformational transition of prion protein; (2) the pK(a)'s of His140 and His177 are approximately 7.0, whereas those of His155 and His187 are < 5.5. 10-ns long molecular dynamics simulations have been performed on five different models, corresponding to the most significant combinations of histidine protonation states. A critical comparison between the available NMR structures and our computational results (1) confirms that His155 and His187 are the residues whose protonation is involved in the conformational rearrangement of huPrP in mildly acidic condition, and (2) shows how their protonation leads to the destructuration of the C-terminal part of HB and to the loss of the last turn of HA that represent the crucial microscopic steps of the rearrangement.

Structure of a Human A-type Potassium Channel Interacting Protein DPPX, a Member of the Dipeptidyl Aminopeptidase Family

It has recently been reported that dipeptidyl aminopeptidase X (DPPX) interacts with the voltage-gated potassium channel Kv4 and that co-expression of DPPX together with Kv4 pore forming alpha-subunits, and potassium channel interacting proteins (KChIPs), reconstitutes properties of native A-type potassium channels in vitro. Here we report the X-ray crystal structure of the extracellular domain of human DPPX determined at 3.0A resolution. This structure reveals the potential for a surface electrostatic change based on the protonation state of histidine. Subtle changes in extracellular pH might modulate the interaction of DPPX with Kv4.2 and possibly with other proteins. We propose models of DPPX interaction with the voltage-gated potassium channel complex. The dimeric structure of DPPX is highly homologous to the related protein DPP-IV. Comparison of the active sites of DPPX and DPP-IV reveals loss of the catalytic serine residue but the presence of an additional serine near the "active" site. However, the arrangement of residues is inconsistent with that of canonical serine proteases and DPPX is unlikely to function as a protease (dipeptidyl aminopeptidase).

Energetic Fitness of Histidine Protonation States in PDB Structures

The energetic fitness of histidine in each of its three protonation states has been investigated for NMR-determined protein structures by using molecular mechanics calculations. The protein structures have been taken from the Protein Data Bank (PDB). For the proteins in the database, we generated all isomers, considering all combinations of protonation forms of each histidine. The energy of each isomer has been determined by conjugate gradient minimization using a well-established all-atom force field. We find that, in general, the isomer reported in the PDB is not the most stable isomer. The statistical distribution of isomer energies minus that of the PDB isomer behaves as though the sequence of the histidine forms reported in the PDB was chosen at random. We also show that our molecular mechanics method is a valid approach to predicting the protonation state of histidines buried in the protein core.

Conformation Dependence of pKa: Ab Initio and DFT Investigation of Histidine

Proton affinity and pKa values of N-formyl-l-histidinamide are found to vary as a function of its backbone and/or side-chain orientation. Proton affinities between the cationic and neutral forms of structurally similar conformers are between −246 and −230 kcal mol-1, while pKa values associated with the same conformers are between 6 and 8. For the neutral-to-anion transition, the following ranges were computed −342 > PA > −350 kcal mol-1 and 18 < pKa < 22. The protonation state of histidines on the surface of a protein depends primarily on the pH. Due to protonation or deprotonation, the side-chain and/or backbone orientation of these histidine residues may undergo considerable changes. Examples are presented and confirmed by ab initio calculations, where proteins were crystallized under various pH conditions, resulting in the same histidine residue to adopt different conformations. Furthermore, a hypothesis is given for a protonation-induced conformational modification of the histidine residue in the cat...

Factors governing the protonation state of Zn-bound histidine in proteins: a DFT/CDM study.

We have performed systematic theoretical studies to elucidate the factors governing the His protonation/deprotonation state in Zn-binding sites, especially those containing the ubiquitous Zn-His-Asp/Glu triad. Specifically, we have addressed the following three questions: (1) How does the transfer of the Zn-bound His imidazole proton to the second-shell Asp/Glu carboxylate oxygen depend on the composition of the other first-shell ligands and the solvent accessibility of the metal-binding site? (2) Can any second-shell ligand with a proton acceptor group such as the backbone carbonyl oxygen also act as a proton acceptor? (3) What is the effect of the Asp/Glu in the Zn-His-Asp/Glu triad on the Zn-bound water protonation state? To address these questions, we used a combination of quantum mechanical and continuum dielectric methods to compute the free energies for deprotonating a Zn-bound imidazole/water in various Zn complexes. The calculations show that whether the Zn-bound His is protonated or deprotonated depends on (1) the solvent accessibility of the metal-binding site, and (2) the Lewis acid ability of Zn, which is indirectly determined by both the first- and the second-shell Zn ligands. The calculations also show that the effect of the Zn-His-Asp/Glu interaction on the nucleophilicity of the Zn-bound water depends on the solvent accessibility of the catalytic Zn site. Furthermore, they show that the Asp/Glu side chain in the Zn-His-Asp/Glu triad can increase the negative charge of its partner, His, and create an anionic hole that may stabilize a cation in buried cavities, provided that the Zn complex is cationic/neutral. The findings of this work are in accord with available experimental data.

Reduction potentials of Rieske clusters: importance of the coupling between oxidation state and histidine protonation state.

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and +150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK(a) values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pK(a), the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q(O) site of the cytochrome bc(1) and b(6)f complexes are discussed.

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.

Tight Binding of Inhibitors to Bovine bc1Complex Is Independent of the Rieske Protein Redox State: CONSEQUENCES FOR SEMIQUINONE STABILIZATION IN THE QUINOL OXIDATION SITE

To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc(1) complex, we measured the binding rate constants (k(1)) for stigmatellin and myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc(1) complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc(1) complex, the k(1) values for stigmatellin ( approximately 2.6 x 10(6) m(-1)s(-1)) and myxothiazol ( approximately 8 x 10(5) m(-1)s(-1)), and the dissociation constant (K(d)) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK(a) approximately 7.6). Reduction of the Rieske protein increased the k(1) value for stigmatellin and decreased the K(d) value for quinone by 50%, without modifying the k(1) for myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.

Histidine pK(a) shifts and changes of tautomeric states induced by the binding of gallium-protoporphyrin IX in the hemophore HasA(SM).

The HasA(SM) hemophore, secreted by Serratia marcescens, binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor, HasR. In HasA(SM), heme is held by two loops and coordinated to iron by two residues, His 32 and Tyr 75. A third residue His 83 was shown recently to play a crucial role in heme ligation. To address the mechanistic issues of the heme capture and release processes, the histidine protonation states were studied in both apo- and holo-forms of HasA(SM) in solution. Holo-HasA(SM) was formed with gallium-protoporphyrin IX (GaPPIX), giving rise to a diamagnetic protein. By use of heteronuclear correlation NMR spectroscopy, the imidazole side-chain (15)N and (1)H resonances of the six HasA(SM) histidines were assigned and their pKa values and predominant tautomeric states according to pH were determined. We show that protonation states of the heme pocket histidines can modulate the nucleophilic character of the two axial ligands and, consequently, control the heme binding. In particular, the essential role of the His 83 is emphasized according to its direct interaction with Tyr 75.

Vibrational Spectra and Ab Initio DFT Calculations of 4-Methylimidazole and Its Different Protonation Forms: Infrared and Raman Markers of the Protonation State of a Histidine Side Chain

The imidazole group of a histidine side chain has four different protonation forms, i.e., the two neutral tautomers (N1- and N3-protonated forms), imidazolium cation, and imidazolate anion. Owing to the presence of these convertible protonation forms, histidine plays important roles in proton-transfer reactions in various enzymes. Vibrational spectroscopy is one of the most powerful methods to study the protonation state of histidine in proteins. For systematic investigation of IR and Raman markers of the protonation state of histidine, we have performed ab initio normal-mode calculations using the density function theory (DFT) method for all of the four protonation forms of 4-methylimidazole (a simple model compound of a histidine side chain) and their N-deuterated analogues. FTIR and Raman spectra of all of these compounds were measured, and the observed bands were assigned according to the calculated frequencies and intensities. Differences in the optimized geometries and changes in the vibrational cou...

2PA039 一酸化炭素結合型西洋ワサビペルオキシダーゼの遠位ヒスチジンのプロトン化状態

2PA039 一酸化炭素結合型西洋ワサビペルオキシダーゼの遠位ヒスチジンのプロトン化状態

Rapid estimation of relative binding affinities of enzyme inhibitors

Computer-assisted molecular modelling (CAMM) comprises a variety of computational methodologies intended to quantitatively or qualitatively describe molecular properties. In some cases, the field has advanced to a state where accurate predictions are possible (e.g. geometric and electronic properties of small molecules). In other cases, however, the properties are complex and require either advances in theory or substantial increases in computational power (e.g. protein folding). One application of CAMM that has received considerable attention over the past two decades entails its use as an aid in drug-design. Ideally, CAMM would provide rapid and accurate prediction of drugtarget binding affinities such that a large and structurally diverse population of potential targets could be evaluated and thereby prioritized prior to chemical synthesis. In reality, methodologies have been advanced that either provide qualitative rank ordering of a large number of molecules in a relatively short period of time or, at the other extreme, generate quantitatively accurate predictions of relative binding affinities for structurally related molecules using substantial computing power. Consequently, techniques that increase speed without greatly compromising accuracy (or vice versa) are of value to drug-discovery programs. Advances in protein crystallography and molecular simulations have greatly aided computer-assisted drug-design paradigms and the accuracy of their binding affinity predictions [1,2]. Methods of inhibitor design range from graphical visualization of the ligand in the binding site to calculation of relative binding affinities using molecular dynamics simulations in conjunction with the TCP approach [3,4]. Figure 1 shows a flowchart employed by drug-discovery groups for structure-based drug-design. Typically, the process begins by generating a working computational model from crystallographic data which includes the development of molecular mechanics parameters for non-standard residues, building any missing segments, assigning the protonation states of histidines, and orientation of carbonyl and amide groups of Asn and Gln amino acid residues based upon neighboring donor/acceptor groups. Inhibitor design is then aided by a variety of visualization tools. For example, hydrophobic and hydrophilic regions of the active site are readily identified by calculating the electrostatic potential at different surface grid points. Frequently, the information gained on the characterization of the active site is supplemented by analyses of ligand conformational energies. These studies are often followed by an estimation of binding affinity which is performed at three different levels of complexity (Fig. 1) depending on computational power, time and resources, namely: (i) qualitative predictions based on docking/ visualization and molecular mechanics calculations; (ii) quantitative predictions based on

Three-dimensional solution structure of human angiogenin determined by 1H,15N-NMR spectroscopy--characterization of histidine protonation states and pKa values.

Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel formation. Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy. 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds. The backbone atomic rms difference from the mean coordinates is 0.067 +/- 0.012 nm and 0.13 nm from the previously determined crystal structure. The side-chain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active-site His residues. The side-chain of His114 adopts two main conformations in solution. In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than His114, with pKa values of 6.65 and 6.05 respectively. The His47 residue is located in an environment very resistant to protonation with a pKa lower than 4.

Structural characterization of the active site of Brucella abortus Cu-Zn superoxide dismutase: a 15N and 1H NMR investigation.

Prokaryotic Cu-Zn superoxide dismutases (SODs) are rare and poorly characterized compared to their eukaryotic counterparts. To better characterize the structure of the prokaryotic enzyme, an NMR investigation of Brucella abortus Cu-Zn SOD in the reduced form was undertaken. The enzyme studied was a recombinant form, expressed in Escherichia coli. The enzyme initially lacked a full complement of Cu and Zn ion. After demetallation and remetallation with a stoichiometric amount of Cu and Zn ion, the specific activity of the recombinant B. abortus Cu-Zn SOD was comparable to the specific activity of the bovine enzyme. The 15N and 1H resonances of seven active site histidine imidazole rings were assigned using two-dimensional NMR methods. A self-consistent set of nuclear Overhauser effects between imidazole ring protons was observed, which was in agreement with the predictions of a model based on the X-ray crystallographic structure of the oxidized bovine enzyme (Tainer, J.A., Getzoff, E. D., Beem, K. M., Richardson, J.S., & Richardson, D.C. (1982) J. Mol. Biol. 160, 181-217). These observations strongly suggest that the structure of the active site of the prokaryotic enzyme is similar to that of the eukaryotic enzyme. Differences in the observed and predicted nuclear Overhauser effects could be ascribed to differences in the oxidation state of the Cu ion (Cu(I) in the reduced B. abortus enzyme and Cu(II) in the oxidized bovine enzyme), as much as they could to the different origins of the enzymes. The NMR data were also compared to a similar 1H NMR study of the human enzyme (Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., & Viezzoli, M. S. (1991) Eur. J. Biochem. 197, 691-697). The pattern of nuclear Overhauser effects and the chemical shifts of corresponding resonances were very similar in 1H NMR spectra of the human and B. abortus enzymes. Significant differences in the chemical shifts or exchange behavior of a few resonances indicated differences in the environments of several histidines in the active sites of reduced B. abortus and human Cu-Zn SODs. This is consistent with the presence of a number of insertions and deletions in the loop regions that make up the active site as indicated by amino acid sequence alignment studies. The tautomeric and protonation states of the active site histidines were also determined in this study, and the results were in agreement with previous studies. The resonances of nitrogen atoms coordinated to metal ions were found to fall between those of protonated and unprotonated nitrogens on histidine imidazoles.