FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022276644> ?p ?o ?g. }

• W2022276644 endingPage "5893" @default.
• W2022276644 startingPage "5886" @default.
• W2022276644 abstract "Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the low-spin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His64 is the endogenous ligand and that Lys67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the ν(Fe-His) stretching frequency appears at 221 cm-1, which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the ν(Fe-CO) stretching frequency was detected at 521 and 494 cm-1 in WT, M69L, and K67T, while only the 494 cm-1 component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm-1 component is attributed to the presence of polar His64. The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His64 and Lys67 comprise a unique distal heme pocket in neuroglobin. Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the low-spin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His64 is the endogenous ligand and that Lys67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the ν(Fe-His) stretching frequency appears at 221 cm-1, which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the ν(Fe-CO) stretching frequency was detected at 521 and 494 cm-1 in WT, M69L, and K67T, while only the 494 cm-1 component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm-1 component is attributed to the presence of polar His64. The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His64 and Lys67 comprise a unique distal heme pocket in neuroglobin. Neuroglobin (Ngb) 1The abbreviations used are: NgbneuroglobinMbmyoglobinHbhemoglobinWTwild-typeWwatts.1The abbreviations used are: NgbneuroglobinMbmyoglobinHbhemoglobinWTwild-typeWwatts. is a new member of the vertebrate globin family, which is expressed predominantly in the brain and nerve tissues (1Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (887) Google Scholar). Although Ngb displays the structural determinants of the globin fold (2Bashford D. Chothia C. Lesk A.M. J. Mol. Biol. 1987; 196: 199-216Crossref PubMed Scopus (443) Google Scholar), the identity in amino acid sequence is low (20-25%) compared with vertebrate hemoglobin (Hb) and myoglobin (Mb) (3Burmester T. Ebner B. Weich B.S. Hankeln T. Mol. Biol. Evol. 2002; 19: 416-421Crossref PubMed Scopus (430) Google Scholar, 4Awenius C. Hankeln T. Burmester T. Biochem. Biophys. Res. Commun. 2001; 287: 418-421Crossref PubMed Scopus (86) Google Scholar, 5Zhang C. Wang C. Deng M. Li L. Wang H. Fan M. Xu W. Meng F. Oian L. Le H. Biochem. Biophys. Res. Commun. 2002; 290: 1411-1419Crossref PubMed Scopus (81) Google Scholar) (Fig. 1). Sequence analyses also show that Ngb is more primitive than Mb (1Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (887) Google Scholar, 6Pesce A. Bolognesi M. Bocedi A. Ascenzi P. Dewilde S. Moens L. Hankeln T. Burmester T. EMBO Rep. 2002; 3: 1146-1151Crossref PubMed Scopus (253) Google Scholar). The function of Ngb is a matter of debate. Ngb is up-regulated in response to hypoxia, and this protects neurons against hypoxic damage (7Sun Y. Jin K. Mao X.O. Zhu Y. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15306-15311Crossref PubMed Scopus (454) Google Scholar). High concentrations (∼100 μm) of Ngb are observed in the retina and its subcellular distribution correlates with the localization of the mitochondria (8Schmidt M. Giessl A. Laufs T. Hankeln T. Wolfrum U. Burmester T. J. Biol. Chem. 2003; 278: 1932-1935Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). These observations point to a role for Ngb in intracellular O2 supply (1Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (887) Google Scholar, 9Trent III, J.T. Watts R.A. Hargrove M.S. J. Biol. Chem. 2001; 276: 30106-30110Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 10Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). However, the low concentration of Ngb in non-pathological brain tissue (in the micromolar range; Ref. 1Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (887) Google Scholar) is hard to reconcile with an O2 storage or carrier function. Indeed, the Hbs of the nervous tissue of several invertebrates (11Dewilde S. Blaxter M. Van Hauwaert M.L. Vanfleteren J. Esmans E.L. Marden M. Griffon N. Moens L. J. Biol. Chem. 1996; 271: 19865-19870Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 12Vandergon T.L. Riggs C.K. Gorr T.A. Colacino J.M. Riggs A.F. J. Biol. Chem. 1998; 273: 16998-17011Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 13Kraus D.W. Colacino J.M. Science. 1986; 232: 90-92Crossref PubMed Scopus (43) Google Scholar), which are known to be involved in O2 transport, occur in much higher concentrations (millimolar concentrations) than Ngb. Only if massive induction occurs can it be envisaged that the concentration of Ngb would reach levels that are sufficient to sustain an aerobic metabolism during temporary hypoxia.Ngb may display as yet unknown enzymatic activities. Cytoglobin, another newly identified member of the globin family (14Kawada N. Kristensen D.B. Asahina K. Nakatani K. Minamiyama Y. Seki S. Yoshizato K. J. Biol. Chem. 2001; 276: 25318-25323Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) ubiquitously expressed in human tissues (3Burmester T. Ebner B. Weich B.S. Hankeln T. Mol. Biol. Evol. 2002; 19: 416-421Crossref PubMed Scopus (430) Google Scholar, 15Geuens E. Brouns I. Flamez D. Dewilde S. Timmermans J.-P. Moens L. J. Biol. Chem. 2003; 278: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), was found to have a peroxidase activity in vitro and to be up-regulated during hepatic inflammation and fibrosis (14Kawada N. Kristensen D.B. Asahina K. Nakatani K. Minamiyama Y. Seki S. Yoshizato K. J. Biol. Chem. 2001; 276: 25318-25323Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 16Asahina K. Kawada N. Kristensen D.B. Nakatani K. Seki S. Shiokawa M. Tateno C. Obara M. Yoshizato K. Biochim. Biophys. Acta. 2002; 1577: 471-475Crossref PubMed Scopus (35) Google Scholar). Ngb could be an O2 sensor protein activating other proteins with regulatory functions (17Kriegl J.M. Bhattacharyya A.J. Nienhaus K. Deng P. Minkow O. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7992-7997Crossref PubMed Scopus (151) Google Scholar, 18Hargrove M.S. Brucker E.A. Stec B. Sarath G. Arredondo-Peter R. Klucas R.V. Olson J.S. Struct. Fold Des. 2000; 8: 1005-1014Abstract Full Text Full Text PDF Scopus (161) Google Scholar). Ferric Ngb, which is generated spontaneously as a result of the rapid auto-oxidation of oxy form, binds exclusively to the GDP-bound form of the α subunit of heterotrimeric G protein (19Wakasugi K. Nakano T. Morishima I. J. Biol. Chem. 2003; 278: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Finally, Ngb might be involved in NO metabolism as has been shown for Mb (20Flögel U. Merx M.W. Godecke A. Decking U.K. Schrader J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 735-749Crossref PubMed Scopus (379) Google Scholar, 21Merx M.W. Flögel U. Stumpe T. Godecke A. Decking U.K. Schrader J. FASEB J. 2001; 15: 1077-1079Crossref PubMed Scopus (90) Google Scholar), a number of flavo-Hbs (22Liu L. Zeng M. Hausladen A. Heitman J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4672-4676Crossref PubMed Scopus (165) Google Scholar), a truncated Hb (23Ouellet H Ouellet Y. Richard C. Labarre M. Wittenberg B. Wittenberg J. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5902-5907Crossref PubMed Scopus (235) Google Scholar), and Ascaris (Nematoda) Hb (24Minning D.M. Gow A.J. Bonaventura J. Braun R Dewhirst M. Goldberg D.E. Stamler J.S. Nature. 1999; 401: 497-502Crossref PubMed Scopus (181) Google Scholar). Whatever its precise role, the function of Ngb is linked to the binding of gaseous ligands.A key feature of Ngb is its six-coordinate heme structure, both in the ferrous and ferric states, with the distal histidine (His64) forming an endogenous ligand (3Burmester T. Ebner B. Weich B.S. Hankeln T. Mol. Biol. Evol. 2002; 19: 416-421Crossref PubMed Scopus (430) Google Scholar, 10Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 25Couture M. Burmester T. Hankeln T. Rousseau D.L. J. Biol. Chem. 2001; 276: 36377-36382Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 26Nistor S.V. Goovaerts E. Van Doorslaer S. Dewilde S. Moens L. Chem. Phys. Lett. 2002; 361: 355-361Crossref Scopus (27) Google Scholar). Amino acid sequence alignments with other globins indicate that the other iron-coordinating ligand is His96 (Fig. 1), and this feature in the ferric state has been confirmed recently by x-ray crystallography (27Pesce A. Dewilde S. Nardini M. Moens L. Ascenzi P. Hankeln T. Burmester T. Bolognesi M. Structure (Lond.). 2003; 11: 1087-1095Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Ngb exhibits high ligand recombination rate constants (kon) and slow dissociation rate constants (koff) for O2 and CO binding, giving rise to a high intrinsic affinity for these exogenous ligands. These ligands must, however, displace the endogenous sixth ligand, resulting in an observed O2 affinity similar to that of Mb (9Trent III, J.T. Watts R.A. Hargrove M.S. J. Biol. Chem. 2001; 276: 30106-30110Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 10Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 25Couture M. Burmester T. Hankeln T. Rousseau D.L. J. Biol. Chem. 2001; 276: 36377-36382Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 28Trent III, J.T. Hargrove M.S. J. Biol. Chem. 2002; 277: 19538-19545Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). In this context, six-coordination is a means of fine-tuning the O2 affinity (10Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Studies of ligand binding over a wide range of temperatures reveal the presence of multiple, intrinsically heterogeneous distal heme pocket conformations in Ngb-CO (17Kriegl J.M. Bhattacharyya A.J. Nienhaus K. Deng P. Minkow O. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7992-7997Crossref PubMed Scopus (151) Google Scholar). This heterogeneity has been attributed to the dissociated His64, which is expected to be situated close to the bound gaseous ligand. However, it has been suggested (17Kriegl J.M. Bhattacharyya A.J. Nienhaus K. Deng P. Minkow O. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7992-7997Crossref PubMed Scopus (151) Google Scholar) that other distal residues compete with His64 for the sixth coordination site.Here we present an extensive resonance Raman analysis of recombinant wild-type (WT) and a series of mutant human Ngbs in their ferric, ferrous, and ferrous-CO bound forms. Through the analysis of various mutants, the endogenous axial ligands have been assigned unequivocally as His64 and His96 in both ferric and ferrous states, as suggested previously. Surprisingly, ligation in the H64V mutant Ngb was found to be pH-dependent, and a further distal residue, Lys67, appears to be situated close to the heme, giving rise to a six-coordinate low-spin structure in both the ferric and ferrous states at alkaline pH. Flash photolysis studies on the CO-bound mutants revealed that both His64 and Lys67 contribute equally to CO binding in the slower phase. Thus, Ngb is revealed to have a distal environment unique among the globins. The role of these residues is discussed in the light of the possible biological functions of Ngb.MATERIALS AND METHODSCloning and Purification of Ngb—To achieve high expression, a gene encoding WT Ngb was synthesized using a modification of the recursive PCR strategy (29Prodromou C. Pearl L.H. Protein Eng. 1992; 5: 827-829Crossref PubMed Scopus (225) Google Scholar, 30Casimiro D.R. Toy-Palmer A. Blake II, R.C. Dyson H.J. Biochemistry. 1995; 34: 6640-6648Crossref PubMed Scopus (58) Google Scholar). Based on the known amino acid sequence of human Ngb (1Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (887) Google Scholar), six forward (f1-f6) and six reverse (r1-r6) primers with lengths ranging from 55 to 60 bases were designed (Fig. 2) and custom-synthesized. Two termination codons (TAA and TGA) were placed in tandem. Each pair of forward and reverse primers overlaps by about 20 bases, enabling chain extension by KOD-Plus-DNA polymerase (Toyobo). The high fidelity of this enzyme enabled perfect match of the synthesized and designed DNA sequences. The full-length PCR product was selectively amplified in a second PCR with a forward primer F which creates tandem XhoI and NdeI sites and a reverse primer R, which generates a BamHI site (Fig. 2). The resultant product was inserted at the XhoI/BamHI site of the plasmid pBluescriptII KS(+) (Stratagene), and the ligation products were used to transform Escherichia coli XL-1 Blue MRF′ (Stratagene). The DNA sequence of the recombinant plasmid product was confirmed with a Li-Cor model 4200S2 DNA sequencer. The QuikChange system (Stratagene) was used to introduce mutations into the Ngb coding sequence, which directed the expression of Ngbs with H64V, K67T, M69L, H96A, and H64V/K67T mutations. The NdeI/BamHI insert was excised from the pBluescript derivatives and ligated into a pET3a-based expression vector (pBEX). The ligation products were introduced into E. coli strain BL21Gold(DE3) (Stratagene). Cell growth and protein purification were performed as described previously (1Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (887) Google Scholar). Proteins were purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis (PAGE) criteria (Fig. 3). Ngb concentrations were evaluated from the heme content which was measured by a pyridine hemochrome assay.Fig. 2Designed gene for human Ngb. Forward (f1-f6, cyan) and reverse (r1-r6, green) primers were custom synthesized. The full-length PCR product was further amplified by the two flanking primers (F and R). Unique restriction sites used for subsequent manipulation of the coding sequence are underlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3SDS-PAGE (left) and absorption spectra (right) of purified H96A and WT Ngb. The absorption spectra were measured in 10 mm sodium phosphate buffer and 0.1 m NaCl (pH 7.0).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Absorption Spectroscopy—The absorption spectra of the purified proteins were recorded on a Beckman DU640 spectrophotometer. pH titration of the ferric Ngb was performed in 10 mm sodium phosphate and 0.1 m NaCl. Aliquots of HCl or NaOH were added to solutions containing ∼10 μm protein. The pH in the cuvette was monitored directly using a microelectrode (Hitachi). The ferrous proteins were also titrated with CO saturated in 10 mm sodium phosphate and 0.1 m NaCl (pH 7.0).Resonance Raman Spectroscopy—Spectra were recorded using a double monochromator (Jasco R-800) with a slit width of 6 cm-1, following excitation by a krypton ion laser (406.7-nm line, Coherent I-302) or by a He-Cd laser (441.6-nm line, Kimmon IK4121R-G). A photomultiplier detector was used (Hamamatsu Photonics, R595), and the frequencies were calibrated with indene. A spinning Raman cell was used throughout the measurements. The samples contained 100 μm protein in the buffers specified in the respective figure legends. Ferrous proteins were prepared by the addition of sodium dithionite, after purging extensively with nitrogen gas. The carbonmonoxy forms were prepared by the addition of sodium dithionite under 1 atm CO. The spectra of the CO forms were obtained with a defocused laser beam.Flash Photolysis—CO rebinding kinetics were monitored at 25 °C using a laser flash photolysis system (Unisoku, Osaka, Japan). After photolysis of ferrous CO-bound Ngb with a 5-ns pulse at 532 nm from a Continuum Q-switched Nd:YAG laser, ligand recombination was followed by monitoring absorbance changes at 420 nm. These experiments were performed with 10 μm Ngbs in 10 mm sodium phosphate and 0.1 m NaCl (pH 7.0) under 1 atm of CO. Selected proteins were photolyzed in 0.1 m Tris-HCl (pH 9.5) under 1 atm of CO.RESULTSHis96—In Fig. 1, the amino acid sequences of selected human globins are compared. Residues identical to those of Ngb are marked in green. The conserved histidines at positions 64 and 96 in Ngb (red) correspond to the distal (E7) and proximal (F8) histidines in Hb and Mb, respectively. The F8 histidine is the sole endogenous axial ligand of the heme iron in the ferric and ferrous states of Hb and Mb, and it is assumed that His96 is the proximal ligand in Ngb. To test this hypothesis, we replaced histidine 96 with alanine (H96A) whose side chain cannot coordinate the iron. This H96A mutant was purified and its mobility in SDS-PAGE was compared with that of WT Ngb (Fig. 3). The absorption spectrum of the ferric WT Ngb is shown (Fig. 3, right) and is essentially the same as that reported previously (10Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). In contrast the H96A mutant is colorless, and the spectrum indicates the complete absence of heme in this mutant. It is concluded that the His96 is essential for heme retention in Ngb, consistent with this residue forming an axial ligand.Lys67 and Met69—It has been proposed that His64 (E7) constitutes the second axial ligand of the heme. In support of this suggestion, the absorption spectrum of the H64L mutant reveals loss of His/His coordination (10Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). However, the Soret peak associated with the ferrous H64L mutant at 423 nm is blue-shifted relative to most five-coordinate Hbs and Mbs (430 nm), and the observed shoulder to this band suggests the presence of two forms, perhaps indicating the presence of an alternative distally coordinated ligand (10Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). To clarify the identity of the distal ligand in WT Ngb, we prepared further mutant Ngbs. Three residues, His64, Lys67, and Met69, are expected to be capable of iron coordination on the distal side of heme (Fig. 1, marked with +). These three residues were mutated to Val, Thr, and Leu, respectively. Thr67 and Leu69 occur in Mb, and the H64V mutant of Mb has been studied extensively (31Quillin M.L. Arduini R.M. Olson J.S. Phillips Jr., G.N. J. Mol. Biol. 1993; 234: 140-155Crossref PubMed Scopus (374) Google Scholar, 32Springer B.A. Sligar S.G. Olson J.S. Phillips Jr., G.N. Chem. Rev. 1994; 94: 699-714Crossref Scopus (719) Google Scholar).In Fig. 4, resonance Raman spectra of ferric WT, M69L, and K67T Ngbs are shown. Resonance Raman spectroscopy is a powerful tool for revealing the oxidation states, spin states, and coordination numbers of heme iron (33Spiro T.G. Li X.-Y. Spiro T.G. Biological Applications of Raman Spectroscopy. Vol. 3. Wiley-Interscience, New York1988: 1-38Google Scholar, 34Kincaid J.R. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. Vol. 7. Academic Press, San Diego, CA2000: 227-291Google Scholar). In the higher frequency region, the ν2, ν38, ν3, and ν4 lines are observed at 1579, 1547, 1504, and 1374 cm-1, respectively. These frequencies are typical of six-coordinate ferric low-spin hemes and similar to those reported previously for the WT Ngb (25Couture M. Burmester T. Hankeln T. Rousseau D.L. J. Biol. Chem. 2001; 276: 36377-36382Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In the lower frequency region, the broad line around 420 cm-1 appears to be slightly affected by the mutation. This line can be assigned to the in-plane bending vibration of the porphyrin vinyl groups (35Hu S. Smith K.M. Spiro T.G. J. Am. Chem. Soc. 1996; 118: 12638-12646Crossref Scopus (463) Google Scholar), and the slight perturbations upon mutation indicate that Met69 and Lys67 are situated close to the heme, though these are not the Fe-coordinating ligands.Fig. 4Resonance Raman spectra of the ferric WT, M69L, and K67T Ngb. From the top: WT, M69L, and K67T. The samples contained 100 μm proteins in 10 mm sodium phosphate buffer and 0.1 m NaCl (pH 7.0). Spectral condition: slit width, 6 cm-1; laser, 406.7 nm at 30 mW.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In Fig. 5, resonance Raman spectra of the ferrous mutants are shown. In the spectrum of WT Ngb, the ν2, ν38, ν3, and ν4 lines at 1580, 1557, 1493, and 1360 cm-1, respectively, indicate the presence of a six-coordinate ferrous low-spin heme (25Couture M. Burmester T. Hankeln T. Rousseau D.L. J. Biol. Chem. 2001; 276: 36377-36382Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Since these frequencies in the spectra of M69L and K67T are identical with those of WT Ngb, Met69 and Lys67 cannot be Fe-coordinating ligands even in the ferrous state. However, the line around 420 cm-1 is again affected by the mutation, suggesting that Met69 and Lys67 are close to the heme in both ferric and ferrous states.Fig. 5Resonance Raman spectra of the ferrous WT, M69L, and K67T Ngb. From the top: WT, M69L, and K67T. The samples contained 100 μm proteins in 10 mm sodium phosphate buffer and 0.1 m NaCl (pH 7.0). Spectral condition: slit width, 6 cm-1; laser, 441.6 nm at 30 mW.View Large Image Figure ViewerDownload Hi-res image Download (PPT)His64—The last candidate for the endogenous axial ligand, His64, was examined. In Table I, the absorption maxima of the ferric mutants are summarized. The wavelengths of these maxima are identical in WT, M69L, and K67T Ngbs, but quite different to those exhibited by the H64V mutant whose Soret maximum occurs at 406 nm at pH 6.0. The spectral features of the H64V Ngb mutant are similar to those of WT Mb at neutral pH. Mb contains a six-coordinate high-spin heme in the ferric state with a water molecule as the sixth ligand of the iron (36Oldfield T.J. Smerdon S.J. Dauter Z. Petratos K. Wilson K.S. Wilkinson A.J. Biochemistry. 1992; 31: 8732-8739Crossref PubMed Scopus (32) Google Scholar).Table IAbsorption maxima of the ferric neuroglobin mutantsProteinpHSoretVisiblenmnmWT7.0413533562M69L7.0413533562K67T7.0413533562H64V6.04065019.5411536H64V/K67T6.04055019.5405501 Open table in a new tab To identify the sixth ligand in H64V, we studied the alkaline transition of this mutant. This transition has been investigated in mammalian Mbs where it is established that the dissociation of one proton equivalent converts a water ligand to a hydroxide ligand at alkaline pH (37Iizuka T. Yonetani T. Adv. Biophys. 1970; 1: 157-182PubMed Google Scholar). As seen in Fig. 6 (upper panel), the absorption spectrum of the ferric H64V Ngb mutant is pH-sensitive in the range pH 6-10. Since one set of isosbestic points was observed, the titration curve was analyzed in terms of a simple equilibrium between the acidic and alkaline forms of the protein. The absorbance changes at the Soret maxima of the two forms were normalized and the molar fraction of the alkaline form was calculated and plotted against pH in Fig. 6 (lower panel). Theoretical curves drawn assuming that the alkaline transition involves one proton equivalent provide a satisfactory fit to the titration data. The apparent pKa value of the alkaline transition in H64V was calculated to be 7.57. This pH-dependent behavior of the H64V is not observed in the WT, K67T, and M69L Ngbs, indicating unequivocally that replacement of His64 is the origin of the alkaline transition. The spectral similarity between the H64V mutant of Ngb and WT Mb and their common pH behavior suggest strongly that each contains an iron-coordinated water in the ferric state at acidic pH.Fig. 6pH titration of ferric H64V Ngb.Upper panel, absorption spectra of the H64V at pH 6.36 (spectrum 1), 7.11 (spectrum 2), 7.57 (spectrum 3), 8.05 (spectrum 4), and 9.13 (spectrum 5). The spectra were measured in 10 mm sodium phosphate and 0.1 m NaCl. Lower panel, molar fraction of alkaline form of H64V. The theoretical curve was drawn assuming one proton equivalent is related to the pH equilibrium.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The pH-dependent characteristics of H64V Ngb were further studied by resonance Raman spectroscopy. At acidic pH, ferric H64V Ngb has a six-coordinate high-spin heme, showing Raman lines at 1561, 1518, 1484, and 1374 cm-1 (Fig. 7). The profile is very similar to that of WT porcine Mb at pH 6.0 (1564, 1514, 1482, and 1372 cm-1) as reported previously (38Uno T. Sakamoto R. Tomisugi Y. Ishikawa Y. Wilkinson A.J. Biochemistry. 2003; 42: 10191-10199Crossref PubMed Scopus (13) Google Scholar). At alkaline pH, the spectra changed drastically, showing lines at 1573, 1540, 1503, and 1374 cm-1, which indicate that H64V contains a six-coordinate low-spin heme at alkaline pH. These frequencies are distinct from those exhibited by Mb at alkaline pH (1587, 1565, 1482, and 1375 cm-1) (38Uno T. Sakamoto R. Tomisugi Y. Ishikawa Y. Wilkinson A.J. Biochemistry. 2003; 42: 10191-10199Crossref PubMed Scopus (13) Google Scholar) and rather closer to those in WT Ngb (Fig. 4). This suggests that the sixth heme ligand of ferric alkaline H64V is not a hydroxide but instead is an endogenous group that is pH-labile.Fig. 7Resonance Raman spectra of the ferric H64V and H64V/K67T Ngb. From the top: H64V at pH 9.5 and at pH 6.4, H64V/K67T at pH 9.5, and at pH 6.4. The samples contained 100 μm proteins in 100 mm sodium phosphate (pH 6.4) or 100 mm Tris-HCl (pH 9.5). Spectral condition: slit width, 6 cm-1; laser, 406.7 nm at 30 mW.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since the structure of ferric H64V Ngb is pH-sensitive, resonance Raman spectra of the ferrous H64V were also measured at acidic and alkaline pH (Fig. 8). At acidic pH, lines were observed at 1564, 1470, and 1356 cm-1, indicating the presence of f" @default.
• W2022276644 created "2016-06-24" @default.
• W2022276644 creator A5000935601 @default.
• W2022276644 creator A5011117420 @default.
• W2022276644 creator A5021407407 @default.
• W2022276644 creator A5026584829 @default.
• W2022276644 creator A5035203295 @default.
• W2022276644 creator A5035765214 @default.
• W2022276644 creator A5077883649 @default.
• W2022276644 creator A5078946015 @default.
• W2022276644 date "2004-02-01" @default.
• W2022276644 modified "2023-10-07" @default.
• W2022276644 title "Residues in the Distal Heme Pocket of Neuroglobin" @default.
• W2022276644 cites W1481105441 @default.
• W2022276644 cites W1547838161 @default.
• W2022276644 cites W1565582771 @default.
• W2022276644 cites W1653986885 @default.
• W2022276644 cites W1886141633 @default.
• W2022276644 cites W1964521153 @default.
• W2022276644 cites W1971498901 @default.
• W2022276644 cites W1974297725 @default.
• W2022276644 cites W1982333430 @default.
• W2022276644 cites W1983181891 @default.
• W2022276644 cites W1989314193 @default.
• W2022276644 cites W1990943605 @default.
• W2022276644 cites W1991430178 @default.
• W2022276644 cites W1991556936 @default.
• W2022276644 cites W2001325871 @default.
• W2022276644 cites W2006542398 @default.
• W2022276644 cites W2007800651 @default.
• W2022276644 cites W2015608678 @default.
• W2022276644 cites W2026669015 @default.
• W2022276644 cites W2031122915 @default.
• W2022276644 cites W2035466780 @default.
• W2022276644 cites W2037967237 @default.
• W2022276644 cites W2039876705 @default.
• W2022276644 cites W2069773991 @default.
• W2022276644 cites W2070664881 @default.
• W2022276644 cites W2072115406 @default.
• W2022276644 cites W2080393204 @default.
• W2022276644 cites W2083960792 @default.
• W2022276644 cites W2088452668 @default.
• W2022276644 cites W2088866723 @default.
• W2022276644 cites W2089107920 @default.
• W2022276644 cites W2094410857 @default.
• W2022276644 cites W2094759883 @default.
• W2022276644 cites W2096700300 @default.
• W2022276644 cites W2098406639 @default.
• W2022276644 cites W2108676680 @default.
• W2022276644 cites W2112880344 @default.
• W2022276644 cites W2115827786 @default.
• W2022276644 cites W2121257509 @default.
• W2022276644 cites W2132397429 @default.
• W2022276644 cites W2142897804 @default.
• W2022276644 cites W2255053361 @default.
• W2022276644 cites W4229570763 @default.
• W2022276644 cites W4236819860 @default.
• W2022276644 cites W99956927 @default.
• W2022276644 doi "https://doi.org/10.1074/jbc.m311748200" @default.
• W2022276644 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14645216" @default.
• W2022276644 hasPublicationYear "2004" @default.
• W2022276644 type Work @default.
• W2022276644 sameAs 2022276644 @default.
• W2022276644 citedByCount "57" @default.
• W2022276644 countsByYear W20222766442012 @default.
• W2022276644 countsByYear W20222766442013 @default.
• W2022276644 countsByYear W20222766442014 @default.
• W2022276644 countsByYear W20222766442015 @default.
• W2022276644 countsByYear W20222766442016 @default.
• W2022276644 countsByYear W20222766442017 @default.
• W2022276644 countsByYear W20222766442018 @default.
• W2022276644 countsByYear W20222766442019 @default.
• W2022276644 countsByYear W20222766442020 @default.
• W2022276644 countsByYear W20222766442022 @default.
• W2022276644 countsByYear W20222766442023 @default.
• W2022276644 crossrefType "journal-article" @default.
• W2022276644 hasAuthorship W2022276644A5000935601 @default.
• W2022276644 hasAuthorship W2022276644A5011117420 @default.
• W2022276644 hasAuthorship W2022276644A5021407407 @default.
• W2022276644 hasAuthorship W2022276644A5026584829 @default.
• W2022276644 hasAuthorship W2022276644A5035203295 @default.
• W2022276644 hasAuthorship W2022276644A5035765214 @default.
• W2022276644 hasAuthorship W2022276644A5077883649 @default.
• W2022276644 hasAuthorship W2022276644A5078946015 @default.
• W2022276644 hasBestOaLocation W20222766441 @default.
• W2022276644 hasConcept C125996951 @default.
• W2022276644 hasConcept C181199279 @default.
• W2022276644 hasConcept C185592680 @default.
• W2022276644 hasConcept C2776217839 @default.
• W2022276644 hasConcept C2776318716 @default.
• W2022276644 hasConcept C2778917026 @default.
• W2022276644 hasConcept C55493867 @default.
• W2022276644 hasConcept C86803240 @default.
• W2022276644 hasConceptScore W2022276644C125996951 @default.
• W2022276644 hasConceptScore W2022276644C181199279 @default.
• W2022276644 hasConceptScore W2022276644C185592680 @default.
• W2022276644 hasConceptScore W2022276644C2776217839 @default.
• W2022276644 hasConceptScore W2022276644C2776318716 @default.

• next