Circular dichroism studies of myoglobin and leghemoglobin

Nicola, Nicos A.; Minasian, E.; Appleby, Cyril A.; Leach, SJ

doi:10.1021/bi00694a019

Cited by 81 publications

(50 citation statements)

References 36 publications

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“…The His-175 --Gly (H175G) mutant was created by site-directed mutagenesis, expressed, purified to homogeneity, and reconstituted with heme as described (6). This purified reconstituted protein was crystallized twice against distilled water and stored as a crystal suspension at 77 K. Protein concentrations were determined from the extinction coefficients determined by pyridine hemochromogens (8,9). In the case of H175G the extinction coefficient was determined for the 280-nm band because of the variability (with pH and temperature) in the intensity and energy of the Soret band (see below).…”

Section: Methodsmentioning

confidence: 99%

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

McRee

Jensen

Fitzgerald

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The crystal structure of the His-175 -) Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (""5%), which, in view ofthe very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.The parameters required of the axial ligands for defining the diverse chemistry of heme enzymes are elusive. Peroxidases often contain a histidine ligand to the iron, whereas cysteine is found in monooxygenases and tyrosine in catalases (1, 2). Cytochrome-c peroxidase (CCP; EC 1.11.1.5) catalyzes the oxidation of cytochrome c (cyt c) by H202 through an intermediate state (ES) that consists of a ferryl (Fe4+=O) heme and a free radical localized on Trp-191 (3). The buried carboxylate of Asp-235 stabilizes this free radical and also accepts a hydrogen bond from the histidine heme ligand, His-175. The resulting Asp/His/metal triad is a common motif in metalloproteins and is reminiscent of the Asp/His/ Ser triad of serine proteases (4,5). In this study, we report the deletion of the histidine iron ligand of CCP to produce a heme enzyme which contains no covalent link to the protein. The facile occupation ofthis cavity by exogenous small molecules demonstrates the potential for producing heme catalysts containing a wide range of unnatural ligands. MATERIALS AND METHODSProtein Expression and Purification. Wild-type and mutant CCP proteins were overexpressed in Escherichia coli BL21(DE3) using the plasmid pT7CCP (6). Wild-type CCP in our laboratory is that with Met-Lys-Thr at the N terminus and containing Ile-53 and Gly-152 (7). The His-175 --Gly (H175G) mutant was created by site-directed mutagenesis, expressed, purified to homogeneity, and reconstituted with heme as described (6). This purified reconstituted protein was crystallized twice against distilled water and stored as a crystal suspension at 77 K. Protein concentrations were determined from the extinction coefficients determined by pyridine hemochromogens (8, 9). In the case of H175G the extinction coefficient was determined for the 280-nm band because of the variability (with pH and temperature) in the intensity and energy of the Soret band (see below).X-Ray Crystallography.t Attempts to obtain crystals of H175G by ...

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Section: Methodsmentioning

confidence: 99%

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

McRee

Jensen

Fitzgerald

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…Such a structure in G. japonicus Mb0 2 , as well as that of soybean leghemoglobin with a more solvent-exposed haem pocket compared with whale myoglobin (Nicola et al 1975;Nicola and Leach 1977), will more easily allow attack by the water molecule and OH-ion on the Fe02 bonding (Shikama et al 1982), resulting in the rapid formation of metMb at neutral and alkaline pH (Table 3).…”

Section: Ink H Kip P K N F V Kit N I a Itt H Kip P H Y F T Kit T I A mentioning

confidence: 99%

Shark Myoglobins. II.Isolation, Characterization and Amino Acid Sequence of Myoglobin from Galeorhinus japonicus

Suzuki¹,

Suzuki²,

Yata³

1985

Aust. Jnl. Of Bio. Sci.

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Native oxymyoglobin (Mb02) was isolated from red muscle of G. japonicus by chromatographic separation from metmyoglobin (metMb) on DEAE-cellulose and the amino acid sequence of the major chain was determined with the aid of sequence homology with that of G. australis. It was shown to differ in amino acid sequence from that of G. australis by 10 replacements, to be acetylated at the amino terminus and to contain glutamine at the distal (E7) residue. It was also shown to have a spectrum very similar to that of mammalian Mb02. However, the pH-dependence for the autoxidation of Mb02 was seen to be quite different from that of sperm whale (Physeter catodon) Mb02. Although the sequence homology between sperm whale and G. japonicus myoglobins is about 40%, their hydropathy profiles were very similar, indicating that they have a similar geometry in their globin folding.

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“…Already, the amino acid sequences of leghaemoglobins from the root nodules of soybean [3], kidney bean [4] and broadbean [5] are known and others are being studied in our laboratory and elsewhere. At the same time, the first X-ray crystal structure determination of a leghaemoglobin has been carried out at a resolution of 5 A, namely that of lupin, while circular dichroism studies have compared the chain folding and the detailed haem environment of a soybean leghaemoglobin with that of sperm whale myoglobin [6,7].…”

Section: Al [ 2 ] )mentioning

confidence: 99%

“…were prepared in a similar manner but not subjected to the final ferricyanide oxidation before use [ll]. Further analytical details and the methods for converting leghaemoglobins to their various liganded states are described elsewhere [7].…”

Section: Preparation Of Leghaemoglobinsmentioning

confidence: 99%

See 1 more Smart Citation

Comparative Structural and Immunochemical Properties of Leghaemoglobins

Hurrell

Nicola

Broughton

et al. 1976

European Journal of Biochemistry

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Circular dichroism studies on leghaemoglobins from snake bean, lupin, serradella and other plants show that, in common with soybean (reported earlier) they have a similar overall polypeptide chain conformation and haem environment and orientation. Immunochemical studies, on the other hand, suggest that the antigenic determinants on the surface of the leghaemoglobins vary considerably.Thus, firstly the a-helix content of the leghaemoglobins as a class is very similar (60-65 x) and approaches that of the myoglobins, secondly, the sign, magnitude and shape of their circular dichroism spectra in the near ultraviolet, Soret and visible regions suggest close similarities in the environment and orientation of a structurally important tryptophan residue and of the haem moiety, and thirdly, there is comparatively weak haem-protein interaction.The extent of immuno cross-reactivity was found to be best demonstrated using the Farr radioimmunoassay procedure. The results were (a) 5 leghaemoglobins from one plant (soybean) crossreacted completely but with varying affinities. (b) The extent of cross reactivity between leghaemoglobins from different plants was compared to that within a single plant; the reaction of antiserum to a soybean leghaemoglobin with a serradella leghaemoglobin was weak, with a snake bean leghaemoglobin still weaker (and incomplete) while lupin leghaemoglobins showed no cross reactivity at all. (c) The "rapid" attenuation of cross reactivity among different plant leghaemoglobins is explicable in terms of the extensive amino acid substitutions which have been demonstrated in the literature and in the present studies. (d) In view of this rapid divergence it is not surprising that sperm whale and horse heart myoglobins showed no cross reactivity with soybean leghaemoglobins.In summary, amino acid substitutions in the leghaemoglobin family are conformationally but not immunochemically conservative.The evolution and genealogy of haemoglobins continues to interest molecular biologists with respect to changes in amino acid sequence, conformation and function. Comparisons of 55 contemporary globins ranging from plant to human haemoglobins show that only two amino acids have remained unchanged during more than 700 million years of evolution. By contrast, the changes in chain conformation have been very small over the same period (see for example, Vainshtein et al. [l]). The rates of evolution of the globin genes have, however, varied considerably, being rapid in the earliest studied stages (up to theinvertebrate-vertebrate ancestor to the chicken-mamma1 ancestor) and slower from that point to the present time (see Goodman et al.[ ] ) .Little is known about the nature and rates of change of globin sequences and conformations prior to the insect and annelid divergences. The availability of many leghaemoglobins from modern plant descendants, however, should permit a study of the earliest ancestry of the globin family. Dozens of such plant species are available and the nodules of a single leguminous plant are know...

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Circular dichroism studies of myoglobin and leghemoglobin

Cited by 81 publications

References 36 publications

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

Shark Myoglobins. II.Isolation, Characterization and Amino Acid Sequence of Myoglobin from Galeorhinus japonicus

Comparative Structural and Immunochemical Properties of Leghaemoglobins

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