Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.     

Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins.

The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate. One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values. Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution. These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein. This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease. The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease. This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group.     

A comparison by 220-MHz NMR of histidine hydronium ion titrations in porcine pancreatic ribonuclease and an extensively deglycosylated derivative.

220-MHz NMR was used to observe the titration behavior of the 5 histidine residues in porcine pancreatic ribonuclease (ribonucleate pyrimidine-nucleotido-2'-transferase (cyclizing), EC 3.1.4.22) and a derivative prepared by removal of 80% of the attached carbohydrate from this glycoprotein. Resonances due to histidine C-2 protons were observed over the full pH range for 3 of the residues; such resonances for the remaining 2 histidine residues broadened out as the pH was increased. Resonances due to histidine C-4 protons were also observed for 2 of the residues. The titration curves for both proteins were identical within experimental error. Resonances were assigned by comparison with histidine NMR titrations in ribonucleases from other species. Histidine 105, immediately adjacent to the site of attachment of a heterosaccharide side chain, has a C-2 proton chemical shift and pK that are insensitive to the large alteration in the bulk of the carbohydrate side chain. The chemical shifts of the C-2 proton of histidine 48 and of the C-4 proton of histidine 80, histidine residues that are close to one another and to another heterosaccharide side chain, show a similar insensitivity. The observations are direct evidence in support of the thesis that the heterosaccharides in porcine ribonuclease project away from the surface of the protein into the solution environment.     

Proton magnetic resonance studies of ribonuclease T1. Assignment of histidine-40 peak and analysis of the active site.

Nuclear magnetic resonances of the C-2 protons of the three histidine residues in ribonuclease T₁ have been studied at 360 MHz as a function of pH to discuss the structure of the active site. Comparison of the order of deuterium exchange of the histidine peaks with tritium incorporation rates into individual histidines of the enzyme leads to the unambigous assignment of one of the C-2 proton peaks to histidne-40. It has been concluded that histidine-40 is in the active site, interacting with a charged group of pK 4.1, which is replaced by the phosphate group of guanosine-3′-monophosphate in the enzyme-inhibitor complex. Histidine-92 is most likely a binding site for the complex, where the existence of a hydrogen bond between N-7 of the inhibitor and the ring NH proton of the histidine is suggested on the basis of NMR data.     

Nuclear magnetic resonance titration curves of histidine ring protons. Conformational transition affecting three of the histidine residues of ribonuclease.

NMR titration curves are reported for the 4 histidine residues of ribonuclease A in sodium acetate and for ribonuclease S in sodium acetate, phosphate, and sulfate solutions. Evidence is presented that the imidazole side chain of histidine residue 48 undergoes a conformational change, probably also involving the carboxyl side chain of aspartic acid residue 14. This group is considered to be responsible for the low pH inflection with pKa 4.2 present in the NMR titration curve of the C-2 proton resonance of histidine 48. The NMR titration curves of the active site histidine residues 12 and 119 also exhibit inflections at low pH values, although there is no carboxyl group within 9 A of the imidazole side chain of histidine residue 12 in the structure of ribonuclease S determined by x-ray crystallography (Wyckoff, H. W., Tsernoglou, D., Hanson, A. W. Knox, J. R., Lee, B., and Richards, F. M. (1970) J. Biol. Chem. 245, 305-328). Curve fitting was carried out on 11 sets of NMR titration data using a model in which the 3 histidine residues 12, 119, and 48 are assumed to be affected by a common carboxyl group. The results obtained indicate that such a model with fewer parameters gives as good a representation of the data as the model in which each histidine residue is assumed to interact separately with a different carboxyl group. Therefore, it is concluded that the ionization of aspartic acid residue 14 is indirectly experienced by the active site histidine residues through the conformational change at histidine 48. A model assuming mutual interaction of the active site histidine residues does not account for the low pH inflections in these curves.     

Nuclear magnetic resonance titration curves of histidine ring protons. The effect of temperature on ribonuclease A.

The ionization constants of 3 of the histidine residues of ribonuclease A have beenobtained at 5 temperatures from the nuclear magnetic resonance titration curves of the imidazole C2 proton resonances. Thermodynamic parameters derived from the ionization constants indicate that histidine residues 105 and 119 are fairly well exposed to solvent, while histidine residue 12 is in a somewhat more restricted environment. Measurements of the low pH inflection present in the titration curve of histidine-12 yield a large negative entropy value, indicating that the group givine rise to this inflection is also buried.     

Characterization of the histidine proton nuclear magnetic resonances of a semisynthetic ribonuclease

The proton magnetic resonance spectrum at 300 MHz of the histidine residues in a semisynthetic derivative of bovine pancreatic ribonuclease (RNase A) has been determined. The derivative RNase 1-118 . 111-124 was prepared by enzymically removing six residues from the COOH terminus of the protein (positions 119-124) and then complementing the inactive RNase 1-118 with a chemically synthesized peptide containing the COOH-terminal 14 residues of ribonuclease (RNase 111-124) [Lin, M.C., Gutte, B., Moore, S., & Merrifield, R.B. (1970) J. Biol. Chem. 245, 5169-5170]. Comparison of the line positions of the C(2)-1H resonances of these residues and of their pH dependence with those reported by other workers has allowed assignment of the resonances to individual residues, as well as the determination of individual pK values for histidine-12, histidine-105, and histidine-119. The assignment of histidine-119 was confirmed by the use of a selectively deuterated derivative. The titration behavior of all four histidine residues is indistinguishable from that observed by others for bovine pancreatic ribonuclease A. Partial dissociation of the noncovalent semisynthetic complex was evident at 30 degrees C, pH 4.0, 0.3 M NaCl; pertinent spectra were analyzed to provide an estimate of the association constant between the component chains under these conditions of 1.9 X 10(3) M-1.     

Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.

The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.     

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments.

The deuterium exchange kinetics of the C(2) protons of the four histidine residues of native bovine pancreatic ribonuclease A have been followed at pH 6.5 and 8.0 by proton magnetic resonance spectroscopy (1H NMR). Comparison of the order of exchange of the histidine peaks with tritium exchange rates into individual histidine residues [Ohe, M., Matsuo, H., Sakiyama, F., and Narita, K. (1974), J. Biochem. (Tokyo) 75, 1197] supports the previous assignment of histidine NMR peaks H(1) and H(4) to histidine-105 and histidine-48 but requires reassignment of peaks H(2) and H(3) to histidine-119 and histidine-12, respectively. Ribonuclease A samples having differentially deuterated histidines have been used to verify the existence of crossover points in the histidine proton magnetic resonance titration curves and to observe the discontinuous titration curve of histidine-48. Proton magnetic resonance peaks have been assigned to the C(4) protons of the four histidine residues of ribonuclease A on the basis of their unit proton areas and by matching their titration shifts with the more readily visible C(2)-H peaks of the histidines. The pK' values derived from the C(4)-H data agree, within experimental limits, with those derived from C(2)-H data. The C(4)-H peaks were assigned to histidine-12, -48, -105, and -119 of ribonuclease A on the basis of their pH dependence, pK' values, shifts of their pK' values in the presence of inhibitor cytidine 3'-phosphate, and by comparison with the assignments of the histidine C(2)-H peaks above.     

Isolation and characterization of a microperoxidase-8 with a modified histidine axial ligand

Microperoxidase-8, Fe(III)MP-8, the heme octapeptide obtained by horse heart cytochrome c digestion, was studied in the presence of H(2)O(2). A modified form of the catalyst was isolated by HPLC and showed a UV/visible spectrum similar to that of Fe(III)MP-8. ESI-MS measurements revealed a 16 Da increase in molecular mass for the modified catalyst when compared to Fe(III)MP-8, suggesting the insertion of an oxygen atom. ESI-MS(2) fragmentation measurements point at oxygen incorporation on the His18 residue of the octapeptide of the modified catalyst. Comparison of the (1)H NMR chemical shifts of the methyl protons of the porphyrin ring of Fe(III)MP-8 and the modified catalyst shows a large shift for especially the 3-methyl and 5-methyl resonances, whereas the other (1)H NMR chemical shifts are almost unaffected. These observations can best be ascribed to a reorientation of the histidine axial ligand. The latter is suggested to be the consequence of an oxygen insertion, possibly on the imidazole ring of His18, thereby corroborating the data obtained by ESI-MS(2). (1)H NMR NOE difference measurements on Fe(III)MP-8 and on the modified catalyst supported the assignment of the H(delta)2 and H(epsilon)1 protons of the His18 imidazole ring. The ring amine proton H(delta)1 could not be detected in both forms of the catalyst. For Fe(III)MP-8 this absence of the H(delta)1 resonance can be ascribed to fast H/D exchange. For the modified catalyst the NMR data are not contradictory, with an oxygen insertion on position delta1 of the His18 imidazole ring with a fast H/D exchanging hydroxyl proton. Together these data converge in suggesting the H(2)O(2) modified catalyst bears a hydroxylated His18 axial ligand. The mechanism that could underlie Fe(III)MP-8 axial histidine hydroxylation is further discussed.     

Correlation of the guanosine exchangeable and nonexchangeable base protons in 13C-/15N-labeled RNA with an HNC-TOCSY-CH experiment

Summary A triple resonance HNC-TOCSY-CH experiment is described for correlating the guanosine imino proton and H8 resonances in ¹³C-/¹⁵N-labeled RNAs. Sequential assignment of the exchangeable imino protons in Watson-Crick base pairs is generally made independently of the assignment of the nonexchangeable base protons. This H(NC)-TOCSY-(C)H experiment makes it possible to unambiguously link the assignment of the guanosine H8 resonances with sequential assignment of the guanosine imino proton resonances. 2D H(NC)-TOCSY-(C)H spectra are presented for two isotopically labeled RNAs, a 30-nucleotide lead-dependent ribozyme known as the leadzyme, and a 48-nucleotide hammerhead ribozyme-RNA substrate complex. The results obtained on these two RNAs demonstrate that this HNC-TOCSY-CH experiment is an important tool for resonance assignment of isotopically labeled RNAs.     

3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

Summary Two new 3D ¹H-¹⁵N-¹³C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the ²J_NC coupling to establish the sequential assignment of backbone NH and ¹⁵N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly ¹⁵N- and ¹³C-labelled ribonuclease T1.     

Modification of the gene 5 DNA unwinding protein with ruthenium

A chemical modification of the gene 5 DNA binding protein (G5BP) from bacteriophage fd was investigated using X-ray diffraction and difference Fourier analysis. The crystalline protein was reacted with pentaammineruthenium (III) trichloride, Ru(NH₃)₅Cl₃, a reagent believed specific for histidine residues and useful in NMR and chemical modification studies of proteins. The major ruthenium site was found by difference Fourier analysis to be 4 Å from histidine 64, the only histidine residue in the molecule. A second bipartite site, believed to be a ruthenium-anion pair, appeared to be salt-bridged to glutamic acid 40 and arginine 16. Indications were present in the difference Fourier results to suggest that the loop containing tyrosine 41 had undergone a slight conformational rearrangement to accommodate this interaction.     

Proton-magnetic-resonance spectroscopic study of the histidine residues of bovine alpha-lactalbumin.

A study of the three histidine residues of bovine alpha-lactalbumin has been made using proton magnetic resonance (PMR) spectroscopy in order to obtain information on their environments in the protein and thereby to test in part the previously proposed structure. PMR titration curves are obtained for the H-4 resonances using difference spectroscopy and for the H-2 resonances and the 1-H-2-H exchange rates of the H-2 protons have been measured. The assignment of resonances to particular histidine residues is achieved by utilising their selective reaction with iodoacetate in conjunction with a PMR study of the carboxymethylation of alpha-N-acetyl-L-histidine. The H-2 and H-4 resonances labelled 1, 2 and 3 starting from the downfield end of the spectrum are assigned to histidine residues 107, 68 and 32 respectively. Their apparent pK values at low ionic strength and 20 degrees C are 5.78, 6.49 and 6.51 respectively. The experimental results on two histidine residues are consistent with the predictions of the proposed structure, which indicate that histidine-68 is an external residue and histidine-32 is partially buried and in the vicinity of aromatic residues. The experimental data on histidine 107 can also be rationalised with less certainty in terms of the proposed structure, which indicates a partially buried residue that may be involved in hydrogen bonding.     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

Proton magnetic resonance spectra of adrenodoxin: features of the aromatic region

This paper presents the first 1H-NMR spectra of the aromatic region of adrenodoxin, a mammalian mitochondrial 2Fe-2S non-heme iron ferredoxin. One-dimensional proton NMR spectra of both reduced and oxidized adrenodoxin were recorded as a function of pH. Resonances due to two of the three histidines of adrenodoxin gave sharp signals in the one-dimensional proton NMR spectra. The pKa values of the resolved histidine resonances in the oxidized protein were 6.64 +/- 0.03 and 6.12 +/- 0.06. These values were unchanged when adrenodoxin was reduced by the addition of sodium dithionite. In addition, the oxidized protein showed a broadened histidine C-2H resonance with a pKa value of approx. 7. This resonance was not apparent in the spectra of the reduced protein. The resonances due to the single tyrosine in adrenodoxin were identified using convolution difference spectroscopy. In addition, a two-dimensional Fourier-transform double quantum filtered (proton, proton) chemical shift correlated (DQF-COSY) spectrum of oxidized adrenodoxin was obtained. The cross peaks of the resonances due to the tyrosine, the four phenylalanines, and two of the three histidines of adrenodoxin were resolved in the DQF-COSY spectrum. Reduction of the protein caused several changes in the aromatic region of the NMR spectra. The resonances assigned to the C2 proton of the histidine with a pKa of 6.6 shifted upfield approx. 0.15 ppm. In addition, when the protein was reduced one of the resonances assigned to a phenylalanine residue with a chemical shift of 7.50 ppm appeared to move downfield to 7.82 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)     

The molecular mechanism of the neutral-to-base transition of human serum albumin. Acid/base titration and proton nuclear magnetic resonance studies on a large peptic and a large tryptic fragment of albumin

In order to obtain a better understanding of the neutral-to-base (N-B) transition of human serum albumin, we performed acid/base titration experiments and 500-MHz 1H NMR experiments on albumin and on a large peptic (residues 1-387) and large tryptic (residues 198-585) fragment of albumin. The acid/base titration experiments revealed that Ca2+ ions induce a downward pK shift of several histidine residues of the peptic (P46) fragment and of albumin. By contrast, Ca2+ has very little influence on the pK of histidine residues of the tryptic (T45) fragment. In albumin, the pH-dependent His C-2 proton resonances, observed with 1H NMR experiments, have been allotted the numbers 1-17. It proved possible to locate these resonances in the P46 and the T45 fragments. A correspondence was found between the number of histidines detected by the acid/base titration and by the 1H NMR experiments. The results of the experiments lead us to conclude that in domain 1 at least the histidines corresponding to the His C-2 proton resonances 1-5 play a dominant role in the N-B transition. The Cu2+-binding histidine residue 3 (resonance 8) of the albumin molecule is not involved in the N-B transition. In addition, we were able to assign His C-2 proton resonance 9 to histidine 464 of the albumin molecule. The role of the N-B transition in the transport and cellular uptake mechanisms of endogenous and exogenous compounds is discussed.     

Automated backbone assignment of labeled proteins using the threshold accepting algorithm

The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, noise, spectral overlap and missing peaks is shown by repeating the assignment with reduced sequential information and increased chemical shift tolerances. The performance of the program on real data is finally demonstrated with automatically picked peak lists of human nonpancreatic synovial phospholipase A₂, a protein with 124 residues.     

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

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)].