Tyrosine Ligand Research Articles

Solution studies of copper(II) complexes as models for the active site in galactose oxidase

The complexation of copper(II)(from the perchlorate) by two tripodal ligands H2L bearing one pyridine and two phenolic arms has been studied in co-ordinating solvents. One of the ligands bears a MeS group at the para position of one of the phenolic moieties. In the presence of triethylamine, only brown dimeric neutral complexes [(CuL)2] have been obtained. In the absence of base, green [Cu(HL)]+ complexes were identified, in which one of the phenolic ligands remains protonated. The brown and the green complexes are reversibly interconverted upon addition of acid or base. Some evidence is given supporting the hypothesis of protonation of the axial tyrosine ligand in the form of galactose oxidase which has been isolated with an exogenous ligand bound to Cu. Electrochemical studies have been performed for the free pro-ligands and for the copper complexes.

Comparative analysis of catalases: spectral evidence against heme-bound water for the solution enzymes

A recent X-ray structural analysis of M. luteus catalase indicates heme-bound H 2O trans to the proximal tyrosinate ligand, a finding in contrast to previous X-ray data reporting a 5-coordinate heme for bovine liver catalase. The presence of heme-bound H 2O, requiring displacement prior to substrate-binding, is likely to be catalytically significant for catalases. We have used magnetic circular dichroism (MCD) spectroscopy, a highly accurate method for assignment of heme spin- and coordination-states, to study native, solution forms of bovine liver, M. luteus, and A. niger catalases. All three enzymes display similar spectral features with the weak (∼5 Δϵ m [moles·cm·Tesla] −1 intensity typical of a 5-coordinate high-spin ferric heme. No evidence for H 2O-ligation, inducing a 6-coordinate heme, occurred upon variation of pH or buffer composition. Therefore, we suggest that the catalytically significant structure of catalases has an unoccupied heme binding site trans to the proximal tyrosinate heme ligand.

Spectroscopic and model studies of astacin, a novel zinc endopeptidase with an endogenous tyrosine ligand

Spectroscopic and model studies of astacin, a novel zinc endopeptidase with an endogenous tyrosine ligand

Ligand interactions with galactose oxidase: mechanistic insights

Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme.

Resonance Raman spectra of horseradish peroxidase and bovine liver catalase compound I species. Evidence for predominant 2A2u pi-cation radical ground state configurations.

The nature of the porphyrin pi-cation radicals in the horseradish peroxidase and bovine liver catalase (BLC) compound I species have been investigated by studying their resonance Raman spectra. A variety of laser excitation and sample interrogation procedures have been employed in order to minimize previously documented problems arising from photoinduced conversions. With Soret band excitation, the spectra obtained for both species resemble that of a compound II-like photoproduct unless the samples are excited with residence times in the microsecond regime with very low (approximately 1 milliwatt) powers. When these precautions are taken, spectra attributable to the compound I species themselves are obtained. The spectrum for horseradish peroxidase compound I is similar to that reported by Paeng and Kincaid (Paeng, K.-J., and Kincaid, J. R. (1988) Am. Chem. Soc. 110, 7913-7915) using a similar approach. Both horseradish peroxidase and BLC compound I exhibit frequency shifts relative to their compound II species that are in the direction observed for model pi-cation radicals with predominant 2A2u character. The magnitudes of these shifts are smaller than those observed for heme models that lack aromatic axial ligands, but agree well with those observed on formation of the compound I analog of N alpha-acetyl microperoxidase-8 that has His as a proximal ligand. This observation is consistent with partial delocalization of the radical density onto the proximal His-170 and Tyr-357 ligands in horseradish peroxidase and BLC, respectively. The strong ligand field provided by these ligands on the proximal side and oxo ligand on the distal side of the heme group is apparently sufficient to reverse the 2A1u radical ground state preference observed for heme-like porphyrin species (e.g. octaethylporphyrins) with weak axial fields. Enhancement of several bands assigned to the Tyr-357 ligand has also been observed for BLC compound I with 406.7-nm excitation. This is attributed either to resonance with a tyrosinate----Fe(IV) charge transfer band or to the coupling provided by radical spin delocalization onto the tyrosinate ligand.

Structure and function of copper-containing proteins: Current Opinion in Structural Biology 1991, 1: 895–904

Insight gained from three-dimensional structures of several cupredoxins has led to site-directed mutagenesis of copper ligands and of adjacent residues relevant to the electron-transfer function of these molecules. Results from these studies have shown that the methionine ligand can be modified and will not perturb function greatly, whereas a conserved hydrophobic patch is important to function. A new X-ray structure of nitrite reductase shows that it a trimer with unexpected sequence and structural similarity to ascorbate oxidase, another multicopper protein of known structure. Each of these multicopper proteins has domain folds like that of the cupredoxins (and superoxide dismutase). The structure of galactose oxidase reveals a three-domain structure which includes one domain with a fold related to the cupredoxin fold, and one with a fold related to that of methylamine dehydrogenase. This structural study also reveals a novel covalent linkage of a cysteine to a tyrosine ligand of the copper center. This linked pair is believed to be the source of a tyrosine radical important to the function of the molecule.

Characteristics in tyrosine coordinations of four hemoglobins M probed by resonance Raman spectroscopy.

Resonance Raman spectra of four hemoglobins (Hbs) M with tyrosinate ligand, that is, Hb M Saskatoon (beta distal His----Tyr), Hb M Hyde Park (beta proximal His----Tyr), Hb M Boston (alpha distal His----Tyr), and Hb M Iwate (alpha proximal His----Tyr), were investigated in order to elucidate structural origins for distinctly facile reducibility of the abnormal subunit of Hb M Saskatoon in comparison with other Hbs M. All of the Hbs M exhibited the fingerprint bands for the Fe-tyrosinate proteins around 1600, 1500, and 1270 cm-1. However, Hb M Saskatoon had the lowest Fe-tyrosinate stretching frequency and was the only one to display the Raman spectral pattern of a six-coordinate heme for the abnormal beta subunit; the others displayed the patterns of a five-coordinate heme. The absorption intensity of Hb M Saskatoon at 600 nm indicated a transition with a midpoint pH at 5.2, whereas that of Hb M Boston was independent of pH from 7.2 to 4.8. The fingerprint bands for the tyrosinate coordination as well as the Fe-tyrosinate stretching band disappeared for Hb M Saskatoon at pH 5.0, and the resultant Raman spectrum resembled that of metHb A, while those bands were clearly observed for Hb M Boston at pH 5.0 and for two Hbs M at pH 10.0. These observations suggest that the unusual characteristics of the heme in the abnormal beta chain of Hb M Saskatoon result from the weak Fe-tyrosinate bond, which allows weak coordination of the proximal histidine, giving rise to the six-coordinate high-spin state at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of soluble cytochromes, ferredoxins and other chromophoric proteins from the halophilic phototrophic bacterium Ectothiorhodospira halophila

A cytochrome c-551 and a pair of ‘high redox-potential’ ferredoxins (iso-high-potential iron-sulfur proteins) were found to be the major soluble electron-transport proteins in Ectothiorhodospira halophila. Smaller amounts of ‘bacterial’ ferredoxin and cytochrome c′ were also observed. With the exception of cytochrome c-551, these proteins are commonly encountered in the purple sulfur bacteria, family Chromatiaceae and less frequently in the purple bacteria, family Rhodospirillaceae. In addition to the cytochromes and ferredoxins, E. halophila synthesizes substantial amounts of a small yellow-colored protein, which has a chromophore spectrally similar to flavins having oxygen, nitrogen or sulfur substituents in place of the 8-methyl group such as roseoflavin and the methanogen cofactor F-420. A purple-colored protein was only partially purified, but it is spectrally similar to iron proteins having a tyrosine ligand, such as transferrin, catechuate dioxygenase, and especially the purple acid phosphatases. Neither the yellow protein nor the purple one has previously been observed in phototrophic bacteria, but may in some way be required for survival in extremely halophilic habitats. The only feature common to halophiles including E. halophila is the very acidic nature of their proteins.

Cobalt(II) as an NMR probe for the investigation of the coordination sites of conalbumin.

The bis and mono cobalt(II) derivatives of conalbumin in the presence of bicarbonate have been prepared. The 1H NMR spectra have been recorded at 60 MHz. Well-shaped isotropically shifted signals in the range -100 to +100ppm have been observed for both the mono and bis cobalt(II) derivatives; besides the intensity, all the isotropically shifted signals for the two derivatives are superimposable, indicating that the two sites are so similar as to be indistinguishable within the resolution of the technique. With the aid of T1 measurements, the signals have been assigned to the protons of two histidine and two tyrosinate ligands. The spectra are consistent with six-coordinated, high-spin cobalt(II) chromophores with two trans tyrosinate moieties at each site.

Cobalt(II) as an NMR probe for the investigation of the coordination sites of conabulmin

Conalbumin and transferrin in the presence of bicarbonate bind metal(III) and also metal(II) ions, although the former oxidation number is preferred. ▪In the absence of oxidizing agents, the cobalt(II) derivatives can be prepared; the conalbumin derivative is particularly inert and both 1:1 and 1:2 cobalt to protein derivatives have been characterized during this research. The 60 MHz proton spectra of cobalt(II) conalbumin recorded in water is shown in Fig. 1. It was obtained using an appropriate pulse sequence (modified DEFT [1]) to suppress the slowly relaxing signals of water and of the diamagnetic protons of the protein. The same sequence was used to evaluate the longitudinal relaxation times of the isotropically shifted signals through a saturation recovery type of experiment. The spectrum shows several well shaped resonances. Within the resolution determined by the linewidth, no difference was observed between the 1:1 and 1:2 derivatives. Assignment of the signals can be attempted through the analysis of the T −1 1 values which are mainly determined by the distance from the paramagnetic center. There is a very broad signal downfield at 100 ppm with a T 1 of 1 ms. Position [2, 3] and T 1 indicate that the signal is due to a histidine Hα. Another quite similar signal is detected when measuring T 1 under the peaks at +67 and +58 ppm. This is a second histidine Hα. The other peaks at 67 and 58 ppm in the same position as peaks found for other tetrahedral and five coordinate cobalt (II) proteins which have been assigned as histidine Hβ protons [2, 3]; even the T 1 values (7−11 ms) are consistent with the assignment. There are then four peaks upfield at −37, −51, −90 and −105 ppm with T 1 values ranging from 2 to 4 ms. They could be the four ortho signals of the two proposed tyrosinate ligands. The signals at −30 and −20 ppm may be assigned to two of the four meta protons of the tyrosinates since the T 1 values of 35 ms indicate a larger proton metal distance. The other two meta signals could be under the intense absorption in the diamagnetic region. Two resonances are still to be assigned, i.e.. that at 31 ppm of intensity 2 and at 19 ppm of intensity 1. Both of them have a T 1 of 11 ms, i.e. they are due to protons relatively close to the metal. The one at 19 ppm could be assigned to a meta signal of a tyrosinate bent in such a way as to give rise to a short proton metal distance. However, a proton of a CH 2 attached to a histidine in a α position seems to fit better with the characteristics of the signal. The assignment of the peak at 31 ppm of intensity two is important even for the chemical implications. It is reasonable to assign the signal to two additional histidine Hβ protons. The spectrum recorded in D 2O after allowing the apo-protein to exchange with D 2O for a week at 35 °C shows that the intensity of the 31 ppm signal is halved, while the signal at +67 ppm disappears. If we assign the 67 ppm signal and one of the 31 ppm signals to histidine NH protons, then at +58 and the residual signal at +31 ppm can be assigned as histidine 4H protons. Based on the present assignment, the NMR spectrum confirms the nature of the ligands in iron transfer proteins and suggests that: i) the histidines bind with the nitrogen close to CH 2; ii)the tyrosinates are in the apical positions of a pseudooctahedral chromophore since only such positions have been found to provide large upfield dipolar shifts.

Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy.

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.

Resonance Raman scattering from uteroferrin, the purple glycoprotein of the porcine uterus.

Uteroferrin, a purple-colored, iron-containing glycoprotein, purified from the uterine fluid of progesterone-stimulated pigs, owes its natural purple color to a broad absorption band centered at 545 nm. Laser excitation within the visible absorption band of uteroferrin results in an intense resonance Raman spectrum which bears a striking resemblance to that reported for Fe(III)-transferrin, the iron transport protein of serum. Excitation profiles for the four resonance-enhanced bands of uteroferrin were obtained from 4579 A to 6741 A, using lines from Ar+ and Kr+ lasers. Each of the profiles have maxima near 545 nm. The spectral similarities of uteroferrin and Fe(III)-transferrin lead to the belief that the Fe(III) binding sites of the two proteins must be, at least in some respects, quite similar. In particular, it is concluded that, as in Fe(III)-transferrin, the metal binding site of uteroferrin contains a tyrosine ligand and that the visible absorption spectrum of uteroferrin results from a phenolate to Fe(III) charge transfer.

A resonance Raman study of substrate and inhibitor binding to protocatechuate-3,4-dioxygenase

Resonance Raman spectra were obtained for complexes of protocatechuate-3,4-dioxygenase with substrate and hydroxybenzoate inhibitors. The data establish metal coordination by these bound species and demonstrate further that tyrosine ligation, present in the resting enzyme, is not altered in the complexes. For the inhibitors, 3-chloro-4-hydroxybenzoate and 3-fluoro-4-hydroxybenzoate, the data are interpreted as indicating iron ligation by the phenolate functionality. For the substrate, 3,4-dihydroxyphenylproprionate, chelation via the o-dihydroxy grouping is proposed. In all three complexes tyrosine ligands present in the resting enzyme are not displaced. The inhibitor scattering intensity was utilized as an internal standard to estimate that two tyrosines are coordinated to the iron at the active site.

ChemInform Abstract: COMPLEXING OF NICKEL(II) BY CYSTEINE, TYROSINE, AND RELATED LIGANDS AND EVIDENCE FOR ZWITTERION REACTIVITY

Chemischer InformationsdienstVolume 6, Issue 28 Organoelement Compounds ChemInform Abstract: COMPLEXING OF NICKEL(II) BY CYSTEINE, TYROSINE, AND RELATED LIGANDS AND EVIDENCE FOR ZWITTERION REACTIVITY J. E. JUN. LETTER, J. E. JUN. LETTERSearch for more papers by this authorROBERT B. JORDAN, ROBERT B. JORDANSearch for more papers by this author J. E. JUN. LETTER, J. E. JUN. LETTERSearch for more papers by this authorROBERT B. JORDAN, ROBERT B. JORDANSearch for more papers by this author First published: July 15, 1975 https://doi.org/10.1002/chin.197528407Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume6, Issue28July 15, 1975 RelatedInformation

Tyrosine ligands to iron in hemerythrin: II. Specification of residues involved

Exposure of Golfingia gouldii hemerythrin to tetranitromethane causes the release of one of the two iron atoms per monomer unit. This loss of iron is associated with the modification of tyrosyl residues, suggesting that tyrosines may be liganded to iron. Tyrosines 18 and 70 in the protein sequence react rapidly with tetranitromethane, forming 3-nitrotyrosine. These nitrotyrosines titrate with normal p K a values. The iron content and circular dichroic spectrum of hemerythrin are unaffected by the nitration of these groups. Hence tyrosines 18 and 70 in the hemerythrin sequence are not ligands to iron. Preliminary evidence suggests that these tyrosines may be in the interfacial areas between subunits in octameric hemerythrin. The remaining three tyrosines per hemerythrin monomer, at positions 8, 67, and 109 in the sequence, also react with tetranitromethane. The product of their reaction is predominantly not 3-nitrotyrosine. Tyrosine 67 forms an ionizable derivative with an absorption maximum at 326–327 nm in basic solution. Little or no intermolecular crosslinking of tyrosines was observed, but the possibility of intramolecular crosslinking was not ruled out. The modification of these tyrosines is accompanied by the loss of one of the two iron atoms per subunit, indicating that one or more of these tyrosines are bound to iron or are in the vicinity of the active site.