The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A_|| = 20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties.     

Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.     

Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin.

Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions. An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45. The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.     

Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence

Fluorescence spectra of wild-type barnase and mutants in which tryptophan and histidine residues have been substituted have been analyzed to give the individual contributions of the three tryptophan residues. The spectrum is dominated by the contribution of Trp-35. The fluorescence intensity varies with pH according to an ionization of a pKa of 7.75. This pKa is close to that previously determined by NMR titration of the C2-H resonances of His-18 as a function of pH (Sali et al., 1989). This histidine residue is close to Trp-94. The pH dependence of the spectrum is abolished when either His-18 or Trp-94 is mutated, and so appears to be caused by the His-18/Trp-94 interaction. The spectral response of this interaction can serve as a probe of the folding pathway and of electrostatic effects within the protein. Changes in the fluorescence spectra on substitution of Trp-94 and His-18 suggest that there is net energy transfer from Trp-71 to Trp-94.     

The surface-associated and secreted MopE protein of Methylococcus capsulatus (Bath) responds to changes in the concentration of copper in the growth medium

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress.     

On the reactivities of the tryptophan residues of human alpha-lactalbumin to 2-hydroxy-5-nitrobenzyl bromide.

The reaction of human alpha-lactalbumin with the tryptophan reagent 2-hydroxy-5-nitrobenzyl bromide has been studied. This protein has 3 tryptophan residues (Trp-60, Trp-104 and Trp-118) all of which are accessible to the reagent at pH 2.7 or 7. Trp-60 of human alpha-lactalbumin is much more reactive than Trp-60 of bovine alpha-lactalbumin (Barman, T. E. (1972) Biochim. Biophys. Acta 257, 297-313). As with bovine alpha-lactalbumin, at pH 2.7, 2-hydroxy-5-nitrobenzyl bromide is specific for tryptophan but at pH 7 His-32 also reacts. When treated with the tryptophan reagent, both alpha-lactalbumins lose their specifier protein activities in the lactose synthase (UDPgalactose:D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) reaction.     

Identification of a copper-repressible C-type heme protein of Methylococcus capsulatus (Bath). A member of a novel group of the bacterial di-heme cytochrome c peroxidase family of proteins

Genomic sequencing of the methanotrophic bacterium, Methylococcus capsulatus (Bath), revealed an open reading frame (MCA2590) immediately upstream of the previously described mopE gene (MCA2589). Sequence analyses of the deduced amino acid sequence demonstrated that the MCA2590-encoded protein shared significant, but restricted, sequence similarity to the bacterial di-heme cytochrome c peroxidase (BCCP) family of proteins. Two putative C-type heme-binding motifs were predicted, and confirmed by positive heme staining. Immunospecific recognition and biotinylation of whole cells combined with MS analyses confirmed expression of MCA2590 in M. capsulatus as a protein noncovalently associated with the cellular surface of the bacterium exposed to the cell exterior. Similar to MopE, expression of MCA2590 is regulated by the bioavailability of copper and is most abundant in M. capsulatus cultures grown under low copper conditions, thus indicating an important physiological role under these growth conditions. MCA2590 is distinguished from previously characterized members of the BCCP family by containing a much longer primary sequence that generates an increased distance between the two heme-binding motifs in its primary sequence. Furthermore, the surface localization of MCA2590 is in contrast to the periplasmic location of the reported BCCP members. Based on our experimental and bioinformatical analyses, we suggest that MCA2590 is a member of a novel group of bacterial di-heme cytochrome c peroxidases not previously characterized.     

The copper responding surfaceome of Methylococccus capsulatus Bath

Proteins on the cellular surface of a bacterium, its surfaceome, are part of the interface between the bacterium and its environment, and are essential for the cells response to its habitat. Methylococcus capsulatus Bath is one of the most extensively studied methane-oxidizers and is considered as a model-methanotroph. The composition of proteins of the surfaceome of M. capsulatus Bath varies with the availability of copper and changes significantly upon only minor changes of copper concentration in the sub-μM concentration range. Proteins that respond to the changes in copper availability include the assumed copper acquisition protein MopE, c-type heme proteins (SACCP, cytochrome c(553o) proteins) and several proteins of unknown function. The most intriguing observation is that multi-heme c-type cytochromes are major constituents of the M. capsulatus Bath surfaceome. This is not commonly observed in bacteria, but is a feature shared with the dissimilatory metal-reducing bacteria. Their presence on the M. capsulatus Bath cellular surface may be linked to the cells ability to efficiently adapt to changing growth conditions and environmental challenges. However, their possible role(s) in methane oxidation, nitrogen metabolism, copper acquisition, redox-reactions and/or electron transport remain(s) at present an open question. This review will discuss the possible significance of these findings.     

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.     

Possible role of the highly conserved amino acids Trp-8 and Pro-13 in the N-terminal segment of the pigment-binding polypeptide LHI alpha of Rhodobacter capsulatus

Trp-8 and Pro-13 of the Rhodobacter capsulatus light-harvesting (LH) I alpha polypeptide are highly conserved among LHI and LHII alpha proteins of several species of the Rhodospirillaceae. Exchange of Trp-8 and Pro-13 to other amino acyl residues similar in structure and/or hydrophobicity indicates that Trp-8 is involved in the insertion of the LHI alpha polypeptide into the intracytoplasmic membrane (ICM). Pro-13, however, seems not to participate in the integration process of the LHI alpha protein but seems to be important for stable insertion of the LHI beta partner protein in the ICM.     

The C-terminal part of the surface-associated protein MopE of the methanotroph Methylococcus capsulatus (Bath) is secreted into the growth medium

A protein with an apparent molecular mass of 46 kDa was detected as the major polypeptide in the culture medium of the biotechnologically important methanotrophic bacterium Methylococcus capsulatus (Bath). The protein cross-reacted with polyclonal antibodies raised against the outer-membrane-associated protein MopE. The antiserum was used to identify a positive clone from a lambda gt11 library. The nucleotide sequence determined for the clone demonstrated that MopE and the secreted protein are encoded by the same gene, and that the secreted protein represents an N-terminally truncated form of MopE. By using monospecific antibodies against MopE in immunogold electron microscopy, the protein was localized at the cell surface and cell periphery. The mopE gene was expressed in Escherichia coli. The MopE protein synthesized was found in the periplasmic space of E. coli. No protein with sequence similarity over the entire length of MopE was detected in the databases, but some sequence similarity to the copper-repressible CorA protein of the methanotroph Methylomicrobium albus (Berson and Lidstrom 1997) was observed for the C-terminal region of MopE.     

A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6

The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.     

Kynurenine metabolism in health and disease

Kynurenine is a small molecule derived from tryptophan when this amino acid is metabolised via the kynurenine pathway. The biological activity of kynurenine and its metabolites (kynurenines) is well recognised. Therefore, understanding the regulation of the subsequent biochemical reactions is essential for the design of therapeutic strategies which aim to interfere with the kynurenine pathway. However, kynurenine concentration in the body may not only be determined by the efficiency of kynurenine synthesis but also by the rate of kynurenine clearance. In this review, current knowledge about the mechanisms of kynurenine production and routes of its clearance is presented. In addition, the involvement of kynurenine and its metabolites in the biology of different T cell subsets (including Th17 cells and regulatory T cells) and neuronal cells is discussed.     

Age-related growth and protein turnover in the thymus of normal and glucocorticoid-treated rats

Possible derivatives of the amino acids tryptophan, tyrosine and histidine were examined as to their effect on protein metabolism in isolated rat hepatocytes. One of the substances tested, kynurenine (a main product of the catabolism of tryptophan), might be a physiological regulator of the lysosomal degradation of endogenous protein, because of the following. (a) Kynurenine decreased the lysosomal (i.e. methylamine-sensitive) pathway of degradation to a much greater extent than its parent amino acid, without interfering with the non-lysosomal pathway. (b) Kynurenine did not appreciably reduce the (lysosomal) degradation of the endocytosed protein asialo-fetuin, or the rate of protein synthesis, indicating a specificity of action. (c) Electron micrographs revealed a reduction in secondary lysosomes due to kynurenine.     

pH dependence of copper geometry, reduction potential, and nitrite affinity in nitrite reductase

Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.     

X-ray crystal structure of the liver X receptor beta ligand binding domain: regulation by a histidine-tryptophan switch

The x-ray crystal structures of the human liver X receptor beta ligand binding domain complexed to sterol and nonsterol agonists revealed a perpendicular histidinetryptophan switch that holds the receptor in its active conformation. Hydrogen bonding interactions with the ligand act to position the His-435 imidazole ring against the Trp-457 indole ring, allowing an electrostatic interaction that holds the AF2 helix in the active position. The neutral oxysterol 24(S),25-epoxycholesterol accepts a hydrogen bond from His-435 that positions the imidazole ring of the histidine above the pyrrole ring of the tryptophan. In contrast, the acidic T0901317 hydroxyl group makes a shorter hydrogen bond with His-435 that pulls the imidazole over the electron-rich benzene ring of the tryptophan, possibly strengthening the electrostatic interaction. Point mutagenesis of Trp-457 supports the observation that the ligand-histidine-tryptophan coupling is different between the two ligands. The lipophilic liver X receptor ligand-binding pocket is larger than the corresponding steroid hormone receptors, which allows T0901317 to adopt two distinct conformations. These results provide a molecular basis for liver X receptor activation by a wide range of endogenous neutral and acidic ligands.     

QM/MM study of the active site of free papain and of the NMA-papain complex.

Hybrid quantum mechanical/molecular mechanical (QM/MM) calculations using restricted and unrestricted Hartree-Fock and B3LYP ab initio (QM) and Amber force field (MM), respectively, have been applied to study the catalytic site of papain in both free and substrate bonded forms. Ab initio geometry optimizations have been performed for the active site of papain and the N-methyl-acetamide (NMA)-papain complex within the molecular mechanical treatment of the protein environment. A covalent tetrahedral intermediate structure could be obtained only when the amide N atom of the substrate molecule was protonated through a proton transfer from the His-159 in the catalytic site. Our results support the previous assumption that a proton transfer from His-159 to the amide N atom of the substrate occurs prior to or concerted with the nucleophilic attack of the Cys-25 sulfur atom to the carbonyl group of the substrate. The electron correlation effect will reduce the proton transfer barrier. Therefore, this proton transfer can be easily observed in the B3LYP/6-31G* calculations. The HF/6-31G* method overestimates the reaction barrier against this proton transfer. The sulfur atom of Cys-25 and the imidazole ring of His-159 are found to be coplanar in the free form of the enzyme. However, the rotation of the imidazole ring of His-159 was observed during the formation of the tetrahedral intermediate. Without the papain environment, the coplanar thiolate-imidazolium ion pair RS-...ImH+ is much less stable than the neutral form of RSH....Im. Within the protein environment, however, the thiolate-imidazolium ion pair becomes more stable than its neutral form by 4.1 and 0.4 kcal/mol in HF/6-31G* and B3LYP/6-31G* calculations, respectively. The barrier of proton transfer from S-H group of Cys-25 to the imidazole ring of His-159 was reduced from 22.0 kcal/mol to 15.2 kcal/mol by the protein environment in HF/6-31G* calculations. This barrier is found to be much smaller (2.5 kcal/mol) in B3LYP/6-31G* calculations.     

Tryptophan phosphorescence and the conformation of liver alcohol dehydrogenase in solution and in the crystalline state

Information on the effects of crystallization upon the structure of liver alcohol dehydrogenase from horse is obtained from a comparison of the phosphorescence properties of its tryptophan residues in solution and in the crystalline state. In the crystalline state the red shift in the phosphorescence spectrum of the solvent-exposed Trp-15 attests to a decreased polarity of its environment consistent with its shielding away from the aqueous solvent probably through its involvement in an intermolecular contact. On the other hand, the triplet-state lifetime of Trp-314 which is buried deeply in the coenzyme-binding domain demonstrates that the flexibility of this region of the macromolecule is unaffected by crystallization; a conclusion supported also by the similarity in the rate of oxygen quenching of its phosphorescence. Given that lattice constraints strongly inhibit large-scale conformational changes these results allow us to identify the average solution structure with the 'open' conformer determined crystallographically.     

Tryptophan metabolism, from nutrition to potential therapeutic applications

Tryptophan is an indispensable amino acid that should to be supplied by dietary protein. Apart from its incorporation into body proteins, tryptophan is the precursor for serotonin, an important neuromediator, and for kynurenine, an intermediary metabolite of a complex metabolic pathway ending with niacin, CO₂, and kynurenic and xanthurenic acids. Tryptophan metabolism within different tissues is associated with numerous physiological functions. The liver regulates tryptophan homeostasis through degrading tryptophan in excess. Tryptophan degradation into kynurenine by immune cells plays a crucial role in the regulation of immune response during infections, inflammations and pregnancy. Serotonin is synthesized from tryptophan in the gut and also in the brain, where tryptophan availability is known to influence the sensitivity to mood disorders. In the present review, we discuss the major functions of tryptophan and its role in the regulation of growth, mood, behavior and immune responses with regard to the low availability of this amino acid and the competition between tissues and metabolic pathways for tryptophan utilization.     

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.