Chloroperoxidase, a janus enzyme

Chloroperoxidase is a versatile fungal heme-thiolate protein that catalyzes a variety of one-electron and two-electron oxidations. We report here that the alkylation of an essential histidine residue showed no effect on the one-electron peroxidations but inhibited two-electron oxidations. The pH profiles of different peroxidative substrates showed optimal activities at varying pH values for the same enzyme. 2-Allylphenol and substituted ortho-phenolics showed efficient peroxidations. Also, substrates excluded from the active site (or with no favorable positioning at the heme center or heme edge) were converted in the peroxidation reaction. While hydrogen peroxide serves as the superior activator in the two-electron oxidations, small alkylhydroperoxides give much better rates for peroxidation reactions. All the above observations indicate that one-electron oxidations are mechanistically quite different from the two-electron oxidations catalyzed by chloroperoxidase. We propose that the peroxidatic substrates interact predominantly outside the heme active site, presumably at the surface of the enzyme.     

The oxidation of naphthalene sulfonate dyes by horse radish peroxidase

Horse radish peroxidase catalyses oxidation of ANS and TNS with hydrogen peroxide. TNS peroxidation may be followed fluorimetrically in the presence of as low as 10^?12m concentrations of the enzyme and permits determination of very low levels of peroxides. Initial rates of peroxidation of ANS and TNS confirmed the general mechanism of peroxidation by HRP. The second-order rate constants for the reduction of HRP compounds I and II were determined. Binding of the substrates to hydrophobic sites of bovine serum albumin or apoperoxidase rendered them inaccessible to the enzyme. While benzhydroxamic acid inhibited the oxidation of dianisidine, it exerted an activating effect on the peroxidation of naphthalene sulfonates. Due to the high reactivity of naphthalene sulfonates, their application as probes in biological systems containing possible traces of peroxidases and peroxides should be interpreted with great caution.     

The effects of pH on the mechanism of hydrogen peroxide and lipid hydroperoxide consumption by myoglobin: a role for the protonated ferryl species

Myoglobin catalyses the breakdown of lipid hydroperoxides (e.g., HPODE) during which the absorption band of the lipid conjugated diene (234 nm) is partially bleached. The constant for this process is strongly pH-dependent (k = 9.5 x 10(-3)s(-1), pH 7: k = 2.3 x 10(-1)s(-1), pH 5). This rate enhancement is not due to acid-induced changes in protein conformation or the involvement of protein-based radical species, as demonstrated by an almost identical pH dependence of the same reaction catalyzed by ferric haemin. The rate constants for ferryl formation and auto-reduction show different pH dependencies, with a pK of 8.3 for ferryl formation and a projected pK of 3.5 for ferryl auto-reduction. The pH dependence for the auto-reduction of the ferryl species is the same as that of the myoblobin catalyzed breakdown of HPODE. We propose that the protonated form of ferryl myoglobin (Fe(4+) - OH(-)) is the reactive species regulating the peroxidatic activity of myoglobin. The protonated ferryl species abstracts an electron from either the protein or porphyrin, allowing fast regeneration of the ferric species. Alkaline conditions stabilize the ferryl species, making myoglobin considerably less reactive towards lipids and lipid hydroperoxides. These findings are significant for understanding myoglobin-induced oxidative stress in vivo and the development of therapies.     

Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H₂O₂ is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H₂O₂.

Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H₂O₂ and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL.

These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.     

Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H₂O₂ is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H₂O₂.

Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H₂O₂ and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL.

These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.     

Electrochemical characterization of lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium

Electrochemical analysis of lignin peroxidase (LiP) was performed using a pyrolytic graphite electrode coated with peroxidase-embedded tributylmethyl phosphonium chloride membrane. The formal redox potential of ferric/ferrous couples of LiP was −126 mV (versus SHE), which was comparable with that of manganese peroxidase (MnP) and horseradish peroxidase (HRP). Yet, only LiP is capable of oxidizing non-phenolic substrates with a high redox potential. Since with decreasing pH, the redox potential increased, an incredibly low pH optimum of LiP as peroxidase at 3.0 or lower was proposed as the clue to explain LiP mechanisms. A low pH might be the key for LiP to possess a high redox potential. The pK_a values for the distal His in peroxidases were calculated using redox data and the Nernst equation, to be 5.8 for LiP, 4.7 for MnP, and 3.8 for HRP. A high pK_a value of the distal His might be crucial for LiP compound II to uptake a proton from the solvent. As a result, LiP is able to complete its catalytic cycle during the oxidation of non-proton-donating substrates. In compensation, LiP has diminished its reactivity toward hydrogen peroxide.     

Oxidation of 2,4-dichlorophenol catalyzed by horseradish peroxidase: characterization of the reaction mechanism by UV-visible spectroscopy and mass spectrometry

The hydrogen peroxide-oxidation of 2,4-dichlorophenol catalyzed by horseradish peroxidase has been studied by means of UV-visible spectroscopy and mass spectrometry in order to clarify the reaction mechanism. The dimerization of 2,4-dichlorophenol to 2,4-dichloro-6-(2,4-dichlorophenoxy)-phenol and its subsequent oxidation to 2-chloro-6-(2,4-dichlorophenoxy)-1,4-benzoquinone together with chloride release were observed. The reaction rate was found to be pH-dependent and to be influenced by the pK(a) value of 2,4-dichlorophenol. The dissociation constants of the 2,4-dichlorophenol/horseradish peroxidase (HRP) adduct at pH 5.5 and 8.5 were also determined: their values indicate the unusual stability of the adduct at pH 5.5 with respect to several adducts of HRP with substituted phenols.     

The mechanism of oxyperoxidase formation from ferryl peroxidase and hydrogen peroxide

Formation of oxyperoxidase from the reaction of ferryl horseradish peroxidase with H2O2 is inhibited by a small amount of tetranitromethane (TNM), a powerful scavenger of superoxide anion radical. The inhibition by TNM, however, does not exceed 35% as the TNM concentration is increased above 5 microM. The stoichiometry of the reaction in the presence of TNM suggests the following equation for TNM-sensitive formation of oxyperoxidase. Ferryl peroxidase + H2O2----(ferric peroxidase + O2- + H+)----oxyperoxidase The kinetic study on the TNM-resistant formation of oxyperoxidase suggests that the displacement of the oxygen with H2O2 takes place at the sixth coordination position at maximal rates of 0.048 and 0.054 s-1 for peroxidases A and C, respectively, at 5 degrees C. The TNM-sensitive and -resistant reactions are concluded to occur in parallel, and both yield oxyperoxidase. In either mechanism, the protonated form of ferryl peroxidase is active and the pK alpha value is 7.1 for peroxidase A and 8.6 for peroxidase C. Oxyperoxidase decomposes spontaneously with a large activation energy (23.0 kcal/mol), and the reaction of ferryl peroxidase with H2O2 reaches a steady level of oxyperoxidase, which depends on pH and the concentration of H2O2.     

Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach

The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25 °C. The activity constants (k_cat) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25 °C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~ 70 °C. The rate of catalysis at temperatures above ~ 70 °C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~ 5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.     

Replacement of the distal glycine 139 transforms human heme oxygenase-1 into a peroxidase

The human heme oxygenase-1 crystal structure suggests that Gly-139 and Gly-143 interact directly with iron-bound ligands. We have mutated Gly-139 to an alanine, leucine, phenylalanine, tryptophan, histidine, or aspartate, and Gly-143 to a leucine, lysine, histidine, or aspartate. All of these mutants bind heme, but absorption and resonance Raman spectroscopy indicate that the water coordinated to the iron atom is lost in several of the Gly-139 mutants, giving rise to mixtures of hexacoordinate and pentacoordinate ligation states. The active site perturbation is greatest when large amino acid side chains are introduced. Of the Gly-139 mutants investigated, only G139A catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, but most of them exhibit a new H(2)O(2)-dependent guaiacol peroxidation activity. The Gly-143 mutants, all of which have lost the water ligand, have no heme oxygenase or peroxidase activity. The results establish the importance of Gly-139 and Gly-143 in maintaining the appropriate environment for the heme oxygenase reaction and show that Gly-139 mutations disrupt this environment, probably by displacing the distal helix, converting heme oxygenase into a peroxidase. The principal role of the heme oxygenase active site may be to suppress the ferryl species formation responsible for peroxidase activity.     

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.     

Oxidation of Benzidine and Its Derivatives by Thyroid Peroxidase

Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3',5,5'-tetramethylbenzidine (TMB). The kinetic characteristics (k(cat) and K(m)) of benzidine (BD), 3,3'-dimethylbenzidine (o-tolidine), 3,3'-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25 degrees C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K(m) values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3', 5, and 5' positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k(cat)/K(m) was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.     

Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.     

Heme-linked ionization of horseradish peroxidase compound II monitored by the resonance Raman Fe(IV)=O stretching vibration

Fe(IV)=O resonance Raman stretching vibrations were recently identified by this laboratory for horseradish peroxidase compound II and ferryl myoglobin. In the present report it is shown that Fe(IV)=O stretching frequency for horseradish peroxidase compound II will switch between two values depending on pH, with pKa values corresponding to the previously reported compound II heme-linked ionizations of pKa = 6.9 for isoenzyme A-2 and pKa = 8.5 for isoenzyme C. Similar pH-dependent shifts of the Fe(IV)=O frequency of ferryl myoglobin were not detected above pH 6. The Fe(IV)=O stretching frequencies of compound II of the horseradish peroxidase isoenzymes at pH values above the transition points were at a high value approaching the Fe(IV)=O stretching frequency of ferryl myoglobin. Below the transition points the horseradish peroxidase frequencies were found to be 10 cm-1 lower. Frequencies of the Fe(IV)=O stretching vibrations of horseradish peroxidase compound II for one set of isoenzymes were found to be sensitive to deuterium exchange below the transition point but not above. These results were interpreted to be indicative of an alkaline deprotonation of a distal amino acid group, probably histidine, which is hydrogen bonded to the oxyferryl group below the transition point. Deprotonation of this group at pH values above the pKa disrupts hydrogen bonding, raising the Fe(IV)=O stretching frequency, and is proposed to account for the lowering of compound II reactivity at alkaline pH. The high value of the Fe(IV)=O vibration of compound II above the transition point appears to be identical in frequency to what is believed to be the Fe(IV)=O vibration of compound X.     

Binding of aromatic donor molecules to lactoperoxidase: proton NMR and optical difference spectroscopic studies

The interaction of aromatic donor molecules with lactoperoxidase (LPO) was studied using 1H-NMR and optical difference spectroscopy techniques. pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with pKa of 6.1) which is presumably a distal histidine. Dissociation constants evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements were found to be an order of magnitude larger than those for binding to horse radish peroxidase (HRP), indicating relatively weak binding of the donors to LPO. The dissociation constants evaluated in presence of excess of I- and SCN- showed a considerable increase in their values, indicating that the iodide and thiocyanate ions compete for binding at the same site. The dissociation constant of the substrate binding was, however, not affected by cyanide binding to the ferric centre of LPO. All these results indicate that the organic substrates bind to LPO away from the ferric center. Comparison of the dissociation constants between the different substrates suggested that hydrogen bonding of the donors with the distal histidine amino acid, and hydrophobic interaction between the donors and the active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the LPO-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative and relatively low compared to those for binding to HRP. The distances of the substrate protons from the ferric center were found to be in the range 9.4-11.1 A which are 2-3 A larger than those reported for the HRP-substrate complexes. These structural informations suggest that the heme in LPO may be more deeply buried in the heme crevice than that in the HRP.     

Chemical and kinetic evidence for an essential histidine in horseradish peroxidase for iodide oxidation.

Horseradish peroxidase (HRP), when incubated with diethylpyrocarbonate (DEPC), shows a time-dependent loss of iodide oxidation activity. The inactivation follows pseudo-first order kinetics with a second order rate constant of 0.43 min-1 M-1 at 30 degrees C and is reversed by neutralized hydroxylamine. The difference absorption spectrum of the modified versus native enzyme shows a peak at 244 nm, characteristic of N-carbethoxyhistidine, which is diminished by treatment with hydroxylamine. Correlation between the stoichiometry of histidine modification and the extent of inactivation indicates that out of 2 histidine residues modified, one is responsible for inactivation. A plot of the log of the reciprocal half-time of inactivation against log DEPC concentration further suggests that only 1 histidine is involved in catalysis. The rate of inactivation shows a pH dependence with an inflection point at 6.2, indicating histidine derivatization by DEPC. Inactivation due to modification of tyrosine, lysine, or cysteine has been excluded. CD studies reveal no significant change in the protein or heme conformation following DEPC modification. We suggest that a unique histidine residue is required for maximal catalytic activity of HRP for iodide oxidation.     

NMR studies of oxyleghemoglobin: assignment of distal histidine proton resonances and evidence for pH-dependent changes in conformation

The resonance of the C-2 proton of the distal histidine has been assigned in the 400 MHz 1H-NMR spectrum of soybean ozyleghemoglobin a. This resonance is subject to a very large ring current shift from the heme and occurs to high field of the residual HO2H peak. The pH dependence was measured from a series of nuclear Overhauser effect difference spectra over a range of pH values. The resonance moves to high field with decreasing pH and reflects titration of a one proton-dissociable group with pK 5.5. Resonances of the heme substituents and distal amino acid side-chains are also sensitive to this titration. Changes in ring-current shifts and nuclear Overhauser effects indicate that a conformational change occurs in the heme pocket upon titration of the pK 5.5 group. We propose that protonation of the distal histidine with pK 5.5 is accompanied by movement of the imidazole ring towards the heme normal. This movement would allow interaction between the ligated oxygen molecule and the protonated distal histidine at acid pH.     

Propyl gallate inhibition of iodide and tetramethylbenzidine oxidation catalyzed by human thyroid peroxidase

The kinetic characteristics (kcat, Km, and their ratio) for oxidation of iodide (I-) at 25 degrees C in 0.2 M acetate buffer, pH 5.2, and tetramethylbenzidine (TMB) at 20 degrees C in 0.05 M phosphate buffer, pH 6.0, with 10% DMF catalyzed by human thyroid peroxidase (HTP) and horseradish peroxidase (HRP) were determined. The catalytic activity of HRP in I- oxidation was about 20-fold higher than that of HTP. The kcat/Km ratio reflecting HTP efficiency was 35-fold higher in TMB oxidation than that in I- oxidation. Propyl gallate (PG) effectively inhibited all four peroxidase processes and its effects were characterized in terms of inhibition constants Ki and the inhibitor stoichiometric coefficient f. For both peroxidases, inhibition of I- oxidation by PG was characterized by mixed-type inhibition; Ki for HTP was 0.93 microM at 25 degrees C. However, in the case of TMB oxidation the mixed-type inhibition by PG was observed only with HTP (Ki = 3.9 microM at 20 degrees C), whereas for HRP it acted as a competitive inhibitor (Ki = 42 microM at 20 degrees C). A general scheme of inhibition of iodide peroxidation containing both enzymatic and non-enzymatic stages is proposed and discussed.     

Structural Basis for Cytochrome c Y67H Mutant to Function as a Peroxidase

The catalytic activity of cytochrome c (cyt c) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt c Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe³⁺ ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H₂O₂ by functioning as an acidic catalyst.     

1H nuclear-magnetic-resonance studies of porcine lutropin and its alpha and beta subunits.

The titration curves of the histidine residues of porcine lutropin and its isolated alpha and beta subunits have been determined by following the pH-dependence of the imidazole C-2 proton resonances. The isolated alpha subunit contains a buried histidine, whose C-2 proton does not exchange with solvent, and which has the unusually low pK of 3.3. In the native hormone all the histidine residues have relatively normal pK values (between 5.7 and 6.2). The four histidine C-2 proton resonances have been assigned to specific residues in the amino-acid sequence, by means of deuterium and tritium exchange experiments on the alpha subunit and its des(92-96) derivative. The histidine with a pK of 3.3 is identified as His-alpha87. The effects of pH on tyrosine and methyl proton resonances show that the titration of His-87 in the isolated alpha subunit is accompanied by a significant conformational change which involves loosening of the protein structure but which is not a normal unfolding transition. The role of conformational changes in the generation of biological activity by subunit association in the glycoprotein hormones is discussed.