Heme-bound tyrosine vibrations in hemoglobin M: Resonance Raman, crystallography, and DFT calculation.

Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or ß subunit contains a ferric heme in the α2ß2 tetramer. Though the ferric subunit cannot bind O2, it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His → tyrosine [Tyr]) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.

Heterogeneity between Two α Subunits of α2ß2 Human Hemoglobin and O2 Binding Properties: Raman, 1H Nuclear Magnetic Resonance, and Terahertz Spectra.

Following a previous detailed investigation of the ß subunit of α2ß2 human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe2+-deoxy/O2)ß(Fe3+) hemoglobin M (Hb M) in which O2 cannot bind to the ß subunit: Hb M Hyde Park (ß92His → Tyr), Hb M Saskatoon (ß63His → Tyr), and Hb M Milwaukee (ß67Val → Glu). In contrast with the ß subunit that exhibited a clear correlation between O2 affinity and Fe2+-His stretching frequencies, the Fe2+-His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α2ß2 tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm-1 region of Hb M Milwaukee, α(Fe2+-deoxy)ß(Fe3+). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe2+-deoxy)ß(Co2+) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O2 affinity by pH change, suggesting no correlation of frequency with O2 affinity for the α subunit. Instead, a new Fe2+-His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O2 affinity increased. The high-frequency counterpart was also observed for a partially O2-bound form, α(Fe2+-deoxy)α(Fe2+-O2)ß(Fe3+)ß(Fe3+), of the present Hb M, consistent with our previous finding that binding of O2 to one α subunit of T structure α2ß2 tetramer changes the other α subunit to the R structure.

Failing blood gas measurement due to methemoglobin forming hemoglobin variants: a case report and review of the literature.

INTRODUCTION: We present a case of an arterial blood gas sample analysis from a 33-year old woman where no oximetry results could be obtained using the Radiometer ABL800 FLEX device. Clinical history of this patient learned that she was carrier of a methemoglobin forming hemoglobin variant type Hyde Park (HbM Hyde Park) and raised the question whether or not this variant could be the cause of the errors obtained during analysis. MATERIALS AND METHODS: A literature search was performed, focusing on methemoglobin forming hemoglobin variants and their influence on oxygenation measurements. An overview of the currently described methemoglobin forming hemoglobin variants is also included. RESULTS AND DISCUSSION: In the presence of dyshemoglobins such as methemoglobin, techniques used to obtain parameters that reflect the patient oxygenation status, such as pulse oximetry and CO-oximetry can be influenced. In these cases, CO-oximetry is the preferred technique because it can compensate for this, in contrast to pulse oximetry. In case of the presence of methemoglobin originating from a hemoglobin variant, it is possible that CO-oximetry data cannot be calculated because the absorbance spectrum of this methemoglobin can differ from regular methemoglobin. Moreover, pulse oximetry devices are actually prone to erroneous results since pulse oximetry data will be calculated in these cases, but unreliable and should be avoided. CONCLUSION: Methemoglobin forming hemoglobin variants are rare genetic mutations. However, they can possibly interfere with the calculation of CO-oximetry values. In these cases, pulse oximetry data should be avoided because they could lead to incorrect medical decisions.

Differences in coordination states of substituted tyrosine residues and quaternary structures among hemoglobin M probed by resonance Raman spectroscopy.

Among the four types of hemoglobin (Hb) M with a substitution of a tyrosine (Tyr) for either the proximal (F8) or distal (E7) histidine in the alpha or beta subunits, only Hb M Saskatoon (betaE7Tyr) assumes a hexacoordinate structure and its abnormal subunits can be reduced readily by methemoglobin (metHb) reductase. This is distinct from the other three M Hbs. To gain new insight into the cause of the difference, we examined the ionization states of E7 and F8 Tyrs by UV resonance Raman (RR) spectroscopy and Fe-O(Tyr) bonding by visible RR spectroscopy. Hb M Iwate (alphaF8Tyr), Hb M Boston (alphaE7Tyr), and Hb M Hyde Park (betaF8Tyr) exhibited two extra UV RR bands at 1,603 cm(-1) (Y8a') and 1,167 cm(-1) (Y9a') arising from deprotonated (ionized) Tyr, but Hb M Saskatoon displayed the UV RR bands of protonated (unionized) Tyr at 1,620 and 1,175 cm(-1) in addition to those of deprotonated Tyr. Evidence for the bonding of both ionization states of Tyr to the heme in Hb M Saskatoon was provided by visible RR spectroscopy. These results indicate that betaE7Tyr of Hb M Saskatoon is in equilibrium between protonated and deprotonated forms, which is responsible for facile reducibility. Comparison of the UV RR spectral features of metHb M with that of metHb A has revealed that metHb M Saskatoon and metHb M Hyde Park are in the R (relaxed) structure, similar to that of metHb A, whereas metHb M Iwate, metHb M Boston and metHb M Milwaukee are in the T (tense) quaternary structure.

Heme structures of five variants of hemoglobin M probed by resonance Raman spectroscopy.

The alpha-abnormal hemoglobin (Hb) M variants show physiological properties different from the beta-abnormal Hb M variants, that is, extremely low oxygen affinity of the normal subunit and extraordinary resistance to both enzymatic and chemical reduction of the abnormal met-subunit. To get insight into the contribution of heme structures to these differences among Hb M's, we examined the 406.7-nm excited resonance Raman (RR) spectra of five Hb M's in the frequency region from 1700 to 200 cm(-1). In the high-frequency region, profound differences between met-alpha and met-beta abnormal subunits were observed for the in-plane skeletal modes (the nu(C=C), nu(37), nu(2), nu(11), and nu(38) bands), probably reflecting different distortions of heme structure caused by the out-of-plane displacement of the heme iron due to tyrosine coordination. Below 900 cm(-1), Hb M Iwate [alpha(F8)His --> Tyr] exhibited a distinct spectral pattern for nu(15), gamma(11), delta(C(beta)C(a)C(b))(2,4), and delta(C(beta)C(c)C(d))(6,7) compared to that of Hb M Boston [alpha(E7)His --> Tyr], although both heme irons are coordinated by Tyr. The beta-abnormal Hb M variants, namely, Hb M Hyde Park [beta(F8)His --> Tyr], Hb M Saskatoon [beta(E7)His --> Tyr], and Hb M Milwaukee [beta(E11)Val --> Glu], displayed RR band patterns similar to that of metHb A, but with some minor individual differences. The RR bands characteristic of the met-subunits of Hb M's totally disappeared by chemical reduction, and the ferrous heme of abnormal subunits was no longer bonded with Tyr or Glu. They were bonded to the distal (E7) or proximal (F8) His, and this was confirmed by the presence of the nu(Fe-His) mode at 215 cm(-1) in the 441.6-nm excited RR spectra. A possible involvement of heme distortion in differences of reducibility of abnormal subunits and oxygen affinity of normal subunits is discussed.

Changes in the abnormal alpha-subunit upon CO-binding to the normal beta-subunit of Hb M Boston: resonance Raman, EPR and CD study.

Heme-heme interaction in Hb M Boston (His alpha 58-->Tyr) was investigated with visible and UV resonance Raman (RR), EPR, and CD spectroscopies. Although Hb M Boston has been believed to be frozen in the T quaternary state, oxygen binding exhibited appreciable co-operativity (n=1.4) and the near-UV CD spectrum indicated weakening of the T marker at pH 9.0. Binding of CO to the normal beta-subunit gave no change in the EPR and visible Raman spectra of the abnormal alpha-subunit at pH 7.5, but it caused an increase of EPR rhombicity and significant changes in the Raman coordination markers as well as the Fe(III)-tyrosine related bands of the alpha-subunit at pH 9.0. The UVRR spectra indicated appreciable changes of Trp but not of Tyr upon CO binding to the alpha-subunit at pH 9.0. Therefore, we conclude that the ligand binding to the beta heme induces quaternary structure change at pH 9.0 and is communicated to the alpha heme, presumably through His beta 92-->Trp beta 37-->His alpha 87.

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study.

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.

Identification of the molecular genetic defect of patients with methemoglobin M-Kankakee (M-Iwate), alpha87 (F8) His --> Tyr: evidence for an electrostatic model of alphaM hemoglobin assembly.

We determined that the molecular defect of 2 patients with hemoglobin (Hb) M-Kankakee [Hb M-Iwate, alpha87 (F8) His --> Tyr] resides in the alpha1-globin gene. The proportion of Hb M observed is higher than that predicted for an alpha1-globin variant. Our evidence suggests that the greater-than-expected proportion of Hb M-Kankakee results from preferential association of the electronegative beta-globin chains with the alpha(M)-globin chains that are more electropositive than normal alpha-globin chains.

Structural and functional properties of human hemoglobins reassembled after synthesis in Escherichia coli.

Human hemoglobin produced in the Escherichia coli coexpression system of Hernan et al. [(1992) Biochemistry 31, 8619-8628] has been transformed into a functionally homogeneous protein whose properties closely approximate those of normal hemoglobin A. Both of the alpha and beta chains of this hemoglobin contain a valine-methionine substitution at position 1 in order to accommodate the difference in specificity of the protein-processing enzymes of procaryotes. Despite extensive purification, functional homogeneity of the E. coli expressed hemoglobin was achieved only by the complete disassembly of the hemoglobin into its component alpha and beta globins and their reassembly in the presence of hemin. The kinetics of CO combination and the thermodynamics of O2 binding and cooperativity of the reassembled alphaV1M-betaV1M hemoglobin closely approximate those of HbA. The alpha globin obtained from the E. coli expressed hemoglobin was also combined with normal human beta chains and hemin to form the alphaV1M variant. The alpha+M variant of HbA, in which the normal N-terminal valine of the alpha chains is preceded by a methionine residue, was prepared by the same procedure. The kinetics of the reactions of CO with the alphaV1M and alpha+M variants are similar to those for HbA. The equilibria of oxygen binding to alphaV1M and HbA are similar whereas alpha+M exhibits a significantly higher oxygen affinity. The three-dimensional structures of alphaV1M and alpha+M offer an explanation for the latter affinity difference. Although the structures of alphaV1M and HbA, which have been determined by X-ray crystallography, are virtually indistinguishable except at the N-terminal residues, that of alpha+M indicates the displacement of a solvent molecule, possibly a chloride ion, from arginine 141alpha. Such an alteration in an anion binding site could result in increased oxygen affinity.

DNA sequence analysis proves Hb M-Milwaukee-2 is due to beta-globin gene codon 92 (CAC-->TAC), the presumed mutation of Hb M-Hyde Park and Hb M-Akita.

Among the causes of congenital methemoglobinemia, Hb M-Milwaukee-2 was one of the earliest described, in a patient who also had Hb E trait. The structure of Hb M-Milwaukee-2 has been elusive. DNA sequence analysis, as here reported, proves that this hemoglobin variant is due to the mutation CAC-->TAC at codon 92 of the beta-globin gene, corresponding to the substitution of tyrosine for histidine. This mutation is identical with that presumed to be the cause of Hb M-Hyde Park and Hb M-Akita. In addition, the DNA mutation of Hb E, GAG-->AAG at codon 26, was confirmed in this case.

Combined mass spectrometric methods for the characterization of human hemoglobin variants localized within alpha T9 peptide: identification of Hb Villeurbanne alpha 89 (FG1) His-->Tyr.

Mutation-induced amino acid exchanges occurring on the large T9 peptide of the alpha-chain of human hemoglobin (residues 62-90) are difficult to identify. Despite their high m/z value (around m/z 3000), collision-induced dissociation spectra of liquid secondary ion mass spectrometrically generated protonated alpha T9 peptides were performed successfully. In parallel electrospray mass spectrometry (MS) was used both to measure the molecular mass of the intact proteins and to determine the number of protonatable sites in the alpha T9 peptides. Peptide ladder sequencing using carboxypeptidase digestions and analysis of the truncated peptides by matrix-assisted laser desorption ionization time-of-flight MS confirmed the interpretation. This set of methods allowed the characterization of three hemoglobin variants, with amino acid exchanges located in the alpha T9 part of the sequence. Two of them, Hb Aztec [alpha 76(EF5) Met-->Thr] and Hb M-Iwate [alpha 87(F8) His-->Tyr] were already known. The third [alpha 89(FG1) His-->Tyr] was novel and named Hb Villeurbanne.

Studies of the oxidation states of hemoglobin M Boston and hemoglobin M Saskatoon in blood by EPR spectroscopy.

The extent of the oxidation of Hemoglobin (Hb) M Saskatoon (beta 63His-->Tyr) and Hb M Boston (alpha 58His-->Tyr) in the patient's blood was determined by measurement of the intensity of EPR signals at g perpendicular = 6.0 for the normal subunits, g1 = 6.7 for the mutant subunits of Hb M Saskatoon and g1 = 6.3 for those of Hb M Boston, respectively. The amounts of reduced mutant subunits were estimated from the EPR signal intensities and the amounts of Hb present as mutant Hb in the blood. About 50% and 76% of mutant subunits in Hb M Boston and Hb M Saskatoon remained reduced in the fresh blood. Gentle shaking of the blood at 37 degrees C for 15 hours in air brought about autoxidation of the normal subunits as well as the mutant subunits of the two Hbs M, indicating that the presence of the mutant subunits facilitated autoxidation of the normal subunits. Possible involvement of NADH-metHb reductase in erythrocytes in maintenance of the reduced mutant subunits of Hb M Saskatoon was discussed.

[Hb M-Iwate [alpha 87 (F8) His-->Tyr]: analysis of the genomic DNA and biosynthesis].

Hb M-Iwate [alpha 87 (F8) His-->Tyr] was identified as the cause of cyanosis in a 21-year-old Japanese female. Amplification and sequencing of the alpha 2- and alpha 1-genes demonstrated the mutation CD87 CAC (His)-->TAC (Tyr) in the alpha 2-gene. Analysis of the in vitro globin biosynthesis in the reticulocytes disclosed a well-balanced beta/alpha synthetic ratio of 1.04 but an unexpectedly low alpha M/total alpha. Although the cause of the lowered alpha M-globin biosynthesis is not yet clear, it might be related to a defect in chain assembly rather than to a modified stability or a reduced amount of the abnormal alpha-globin mRNA.

Determination of Fe-CO geometry in the subunits of carbonmonoxy hemoglobin M Boston using femtosecond infrared spectroscopy.

We have undertaken ultrafast infrared (IR) spectroscopic studies in order to elucidate the geometry of bound CO in the alpha and beta subunits of hemoglobin (Hb) M Boston 13CO. Hb M Boston is a mutant human Hb in which the distal histidine in the alpha subunits is replaced by a tyrosine. The IR absorptions of bound 13CO fall at 1925 cm-1 for the alpha subunits and 1907 cm-1 for the beta subunits. Despite a difference of nearly 20 cm-1 in these peaks, the measured anisotropies of the bound 13CO depletions following 30% photolysis are nearly identical, with values of -0.142 +/- 0.002 obtained for the alpha subunits and -0.140 +/- 0.003 obtained for the beta subunits. These translate to values of 20 degrees +/- 1 degree and 21 degrees +/- 1 degree for the values of the average angles between the CO bond and the normal to the heme planes in the alpha and beta subunits, respectively. Our present results and the work of previous investigators [Nagai, M., Yoneyama, Y., & Kitagawa, T. (1991) Biochemistry 30, 6495-6503] suggest that a change in the polar interactions of the bound CO with the heme pocket environment upon substitution of tyrosine for the distal histidine and a less bent structure for the Fe-C-O unit in the alpha subunits are responsible for the difference in the bound CO absorption frequencies in the alpha and beta subunits. A spectrum of the depletion of the bound 13CO peaks following photolysis indicates that both subunits photodissociate CO with the same quantum yield and neither subunit exhibits significant recombination within 1 ns.

Concise review: methemoglobinemia.

The ferrous iron of hemoglobin is exposed continuously to high concentrations of oxygen and, thereby, is oxidized slowly to methemoglobin, a protein unable to carry oxygen. To restore hemoglobin function, methemoglobin (ferrihemoglobin) must be reduced to hemoglobin (ferrohemoglobin). Under physiological conditions, methemoglobin reduction is accomplished mainly by red cell NADH-cytochrome b5 reductase (NADH-methemoglobin reductase) so efficiently that there is insignificant amounts of methemoglobin in the circulating blood. However, should methemoglobin formation be increased--e.g., due to the presence of oxidant drugs, or an abnormal methemoglobin not amenable to reduction (hemoglobin M), or a deficiency in red cell cytochrome b5 reductase--methemoglobinemia will result. Most methemoglobinemias have no adverse clinical consequences and need not be treated. Under certain conditions, such as exposure to large amounts of oxidant or in young infants, rapid treatment is necessary. In hereditary cytochrome b5 deficiency, treatment is often directed at improving the poor cosmetic effect of persistent cyanosis with the minimum amount of drugs to give satisfactory clinical results.

A second observation of the fetal methemoglobin variant Hb F-M-Fort Ripley or alpha 2G gamma 2(92)(F8)His----Tyr.

We have identified a second baby with the fetal methemoglobin F-M-Fort Ripley. It was observed in a Caucasian infant from Canada; at least eleven additional members of that family were known to have had a neonatal cyanosis similar to that seen in the propositus and in a previously described baby (2). Sequencing of amplified DNA that included (part of) the G gamma gene greatly facilitated the characterization. The G gamma X chain was readily isolated by reversed phase high performance liquid chromatography; its quantity was approximately 12.5% of total gamma. Interestingly, the baby also carried the A gamma T mutation on one chromosome, either in cis or in trans to the G gamma X mutation. Hb F-M-Fort Ripley could be isolated in reasonably pure form by DEAE-cellulose chromatography. The isolated Hb FX was unstable, had spectral changes characteristic for the M-hemoglobins, while its methemoglobin derivative reacted rapidly with cyanide. Oxygen affinity data could not be obtained. It is suggested that the formation of a rather large amount (approximately 25%) of mixed hybrids (alpha 2G gamma X.gamma) with low oxygen affinity is the main cause for the occurrence of the neonatal cyanosis.