In order to assess the relative importance of polar versus steric interactions, infrared spectra and overall CO binding properties were measured at room temperature for 41 different recombinant myoglobins containing mutations at His64(E7), Val68(E11), Phe43(CD1), Arg45(CD3), Phe46(CD4), and Leu29(B10). The results were compared to the crystal structures of wild-type, Phe29, Val46, Ala68, Phe68, Gln64, Leu64, and Gly64 sperm whale CO-myoglobin and that of Thr68 pig CO-myoglobin. As observed in several previous studies, replacement of the distal histidine (His64) with aliphatic amino acids results in the appearance of a single IR band in the 1960-1970-cm-1 region and in large increases in CO affinity (KCO). More complex behavior is observed for Gly, Ala, Gln, Met, and Trp substitutions at position 64, but in each case there is a net increase in the intensity of this high-frequency component. Replacement of Val68 with Ala, Leu, Ile, and Phe produces little effect on the IR spectrum, whereas these mutations cause 20-fold changes in KCO, presumably due to steric effects. Replacement of Val68 with Thr decreases KCO 4-5-fold, whereas the position of the major IR band increases from 1945 to 1961 cm-1. Replacement of Val68 with Asn also causes a large decrease in KCO, but in this case, the peak position of the major IR band decreases from 1945 to 1916 cm-1. Nine replacements were made in the CD corner at positions 43, 45, and 46. All of the resultant mutants show increased stretching frequencies that can be correlated with movement of the imidazole side chain of His64 away from the bound ligand. All five substitutions at position 29 cause changes in the IR spectra. The Leu29-->Phe mutation had the largest effect, producing a single band centered at 1932 cm-1. Together these data demonstrate that there is little direct correlation between affinity, vCO, and Fe-C-O geometry. The major factor governing vCO appears to be the electrostatic potential surrounding the bound ligand and not steric hindrance. The presence of positive charges from proton donors, such as N epsilon of His64 and N delta of Asn68, cause a decrease in the bond order and stretching frequency of bound CO. In contrast, the negative portion of the Thr68 dipole points directly toward the bound ligand and increases the C-O bond order and stretching frequency. Movement of His64 away from the bound ligand or replacement of this residue with aliphatic amino acids prevents hydrogen-bonding interactions, causing vCO to increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue. In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O2]. The bimolecular rate constants for NO-induced oxidation of MbO2 and HbO2 are large (k.ox,NO = 30-50 microM-1 s-1 for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb. Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe(2+)-N=O or with Fe(2+)-O-O delta- to produce Fe(3+)-OH2 and nitrate. Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO2. The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O2 carriers. This approach has been explored by examining the effects of Phe(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.

Most recent experiments have indicated that distal pocket polarity rather than steric hindrance is the major factor governing the distribution of FeCO stretching frequencies (ν C-O , ν Fe-CO ) in myoglobins and hemoglobins. Hydrogen bonding and other polar interactions have also been shown to play a key role in regulating O 2 and CO binding. To quantify the effects of polarity on ν C-O , ν Fe-CO , and ligand binding, we calculated electrostatic potential field distributions in the distal pockets of 18 different mutants and two wild-type forms of recombinant pig and sperm whale MbCO. The results were obtained using linearized Poisson-Boltzmann methods with coordinates from high-resolution structures determined experimentally by X-ray crystallography. The computed potential fields at the ligand atoms vary from +30 to -12 kcal/mol depending on the protein structure at the distal site. The electrostatic fields correlate inversely with ν C-O and directly with ν Fe-CO . In all our calculations, the distal histidine is modeled as the neutral N -H tautomer, regardless of which ferrous ligand is bound. If the neutral Nδ-H tautomer is used, the computed potentials at the bound ligand atoms are uniformly negative and show no correlation with ν C-O , ν Fe-C , and any ligand binding parameters. Although calculated using primarily MbCO structures, there is a linear, inverse relationship between the electrostatic field at the ligand binding site and the logarithm of the rate constant for O 2 dissociation. As a result, high O 2 affinity can be predicted semiquantitatively from a large positive potential field or from an experimentally low value of ν C-O . Thus, the stretching frequency of bound CO serves as an empirical voltmeter that can be used to measure the polarity of the distal pocket and to predict the extent of electrostatic stabilization of bound O 2 .

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The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.