Assignment of proton NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

Abstract

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].

Temperature dependence of histidine ionization constants in myoglobin

“…2 indicate a preference to form the NE2H tautomer of the neutral imidazole. His-48 is sensitive to a high pH event, taken to be hemic acid dissociation (Carver and Bradbury, 1984;Cocco et al, 1992). The temperature dependence of this second transition is in agreement with this assumption.…”

“…The protein undergoes hemic acid dissociation (loss of a hydrogen ion by the liganded water) near pH 8.8 (George and Hanania, 1952;Brunori et al, 1968). The high-to-low-spin alkaline transition is observed in the proton chemical shifts of many residues, including a few histidines (Carver and Bradbury, 1984;Cocco et al, 1992). When this transition is detected in a chemical-shift-versus-pH profile, a second Henderson-Hasselbalch term is necessary.…”

Analysis of zinc binding sites in protein crystal structures

“…The existence of H-bonds between zinc ligands and protein residues that are not part of the zinc primary coordination shell has been studied by a number of workers (Christianson, 1991;Kiefer et al, 1995;Lesburg & Christianson, 1995). For example, a carboxylate-His H-bond is believed to increase the basicity and ligand strength of the His and arrange it correctly for interaction with the metal (Argos et al, 1978;Carver & Bradbury, 1984;Perutz et al, 1985).…”

Ferrocyanide − a novel catalyst for oxymyoglobin oxidation by molecular oxygen

“…In the pH 5–8 range investigated, sperm whale Mb (pI 8.3) is positively charged, whereas the overall charge of horse Mb (pI 7.4) and pig Mb (pI 7.2) changes from positive to negative values. In horse myoglobin, in contrast to the sperm whale protein, the solvent‐exposed His12 is replaced by Gln and, in pig myoglobin, three residues, His12, His113 and His116, are also replaced by Gln [9,10]. Another 19 replacements in these native proteins are identical or homologous except for the replacement of positively charged Lys87(F2) of sperm whale and horse Mb by neutral Thr87 in pig Mb.…”