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.

Structural origin of cooperativity in human hemoglobin: a view from different roles of α and ß subunits in the α2ß2 tetramer.

This mini-review, mainly based on our resonance Raman studies on the structural origin of cooperative O2 binding in human adult hemoglobin (HbA), aims to answering why HbA is a tetramer consisting of two α and two ß subunits. Here, we focus on the Fe-His bond, the sole coordination bond connecting heme to a globin. The Fe-His stretching frequencies reflect the O2 affinity and also the magnitude of strain imposed through globin by inter-subunit interactions, which is the origin of cooperativity. Cooperativity was first explained by Monod, Wyman, and Changeux, referred to as the MWC theory, but later explained by the two tertiary states (TTS) theory. Here, we related the higher-order structures of globin observed mainly by vibrational spectroscopy to the MWC theory. It became clear from the recent spectroscopic studies, X-ray crystallographic analysis, and mutagenesis experiments that the Fe-His bonds exhibit different roles between the α and ß subunits. The absence of the Fe-His bond in the α subunit in some mutant and artificial Hbs inhibits T to R quaternary structural change upon O2 binding. However, its absence from the ß subunit in mutant and artificial Hbs simply enhances the O2 affinity of the α subunit. Accordingly, the inter-subunit interactions between α and ß subunits are nonsymmetric but substantial for HbA to perform cooperative O2 binding.

Roles of Fe-Histidine bonds in stability of hemoglobin: Recognition of protein flexibility by Q Sepharose.

Using various mutants, we investigated to date the roles of the Fe-histidine (F8) bonds in cooperative O2 binding of human hemoglobin (Hb) and differences in roles between α- and ß-subunits in the α2ß2 tetramer. An Hb variant with a mutation in the heme cavity exhibited an unexpected feature. When the ß mutant rHb (ßH92G), in which the proximal histidine (His F8) of the ß-subunit is replaced by glycine (Gly), was subjected to ion-exchange chromatography (Q Sepharose column) and eluted with an NaCl concentration gradient in the presence of imidazole, yielded two large peaks, whereas the corresponding α-mutant, rHb (αH87G), gave a single peak similar to Hb A. The ß-mutant rHb proteins under each peak had identical isoelectric points according to isoelectric focusing electrophoresis. Proteins under each peak were further characterized by Sephadex G-75 gel filtration, far-UV CD, 1H NMR, and resonance Raman spectroscopy. We found that rHb (ßH92G) exists as a mixture of αß-dimers and α2ß2 tetramers, and that hemes are released from ß-subunits in a fraction of the dimers. An approximate amount of released hemes were estimated to be as large as 30% with Raman relative intensities. It is stressed that Q Sepharose columns can distinguish differences in structural flexibility of proteins having identical isoelectric points by altering the exit rates from the porous beads. Thus, the role of Fe-His (F8) bonds in stabilizing the Hb tetramer first described by Barrick et al. was confirmed in this study. In addition, it was found in this study that a specific Fe-His bond in the ß-subunit minimizes globin structural flexibility.

A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy.

Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O2 or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the ß subunits. However, these changes are not observed in the isolated ß chain (ß4 homotetramer) and, therefore, are associated with the α-ß inter-subunit interactions. For the communications between α and ß subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O2 binding mechanism of Hb A.

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.

Interrelationship among Fe-His Bond Strengths, Oxygen Affinities, and Intersubunit Hydrogen Bonding Changes upon Ligand Binding in the ß Subunit of Human Hemoglobin: The Alkaline Bohr Effect.

Regulation of the oxygen affinity of human adult hemoglobin (Hb A) at high pH, known as the alkaline Bohr effect, is essential for its physiological function. In this study, structural mechanisms of the alkaline Bohr effect and pH-dependent O2 affinity changes were investigated via 1H nuclear magnetic resonance and visible and UV resonance Raman spectra of mutant Hbs, Hb M Iwate (αH87Y) and Hb M Boston (αH58Y). It was found that even though the binding of O2 to the α subunits is forbidden in the mutant Hbs, the O2 affinity was higher at alkaline pH than at neutral pH, and concomitantly, the Fe-His stretching frequency of the ß subunits was shifted to higher values. Thus, it was confirmed for the ß subunits that the stronger the Fe-His bond, the higher the O2 affinity. It was found in this study that the quaternary structure of α(Fe3+)ß(Fe2+-CO) of the mutant Hb is closer to T than to the ordinary R at neutral pH. The retained Aspß94-Hisß146 hydrogen bond makes the extent of proton release smaller upon ligand binding from Hisß146, known as one of residues contributing to the alkaline Bohr effect. For these T structures, the Aspα94-Trpß37 hydrogen bond in the hinge region and the Tyrα42-Aspß99 hydrogen bond in the switch region of the α1-ß2 interface are maintained but elongated at alkaline pH. Thus, a decrease in tension in the Fe-His bond of the ß subunits at alkaline pH causes a substantial increase in the change in global structure upon binding of CO to the ß subunit.

Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or ß Subunits: Circular Dichroism, (1) H NMR, and Resonance Raman Studies.

Native human adult hemoglobin (Hb A) has mostly normal orientation of heme, whereas recombinant Hb A (rHb A) expressed in E. coli contains both normal and reversed orientations of heme. Hb A with the normal heme exhibits positive circular dichroism (CD) bands at both the Soret and 260-nm regions, while rHb A with the reversed heme shows a negative Soret and decreased 260-nm CD bands. In order to examine involvement of the proximal histidine (His F8) of either α or ß subunits in determining the heme orientation, we prepared two cavity mutant Hbs, rHb(αH87G) and rHb(ßH92G), with substitution of glycine for His F8 in the presence of imidazole. CD spectra of both cavity mutant Hbs did not show a negative Soret band, but instead exhibited positive bands with strong intensity at the both Soret and 260-nm regions, suggesting that the reversed heme scarcely exists in the cavity mutant Hbs. We confirmed by (1) H NMR and resonance Raman (RR) spectroscopies that the cavity mutant Hbs have mainly the normal heme orientation in both the mutated and native subunits. These results indicate that the heme Fe-His F8 linkage in both α and ß subunits influences the heme orientation, and that the heme orientation of one type of subunit is related to the heme orientation of the complementary subunits to be the same. The present study showed that CD and RR spectroscopies also provided powerful tools for the examination of the heme rotational disorder of Hb A, in addition to the usual (1) H NMR technique. Chirality 28:585-592, 2016. © 2016 Wiley Periodicals, Inc.

Utility of heme analogues to intentionally modify heme-globin interactions in myoglobin.

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and ß Subunits in the α2ß2 Tetramer.

Human hemoglobin (Hb), which is an α2ß2 tetramer and binds four O2 molecules, changes its O2-affinity from low to high as an increase of bound O2, that is characterized by 'cooperativity'. This property is indispensable for its function of O2 transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and ß subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and ß subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or ß subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O2-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O2-binding with no cooperativity, indicating the coexistence of two independent hemes with different O2-affinities. In contrast, rHb(ßH92G), in which only the Fe-His in the ß subunits is replaced by Fe-imidazole, gave a simple high-affinity O2-binding curve with no cooperativity. Resonance Raman, 1H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O2-binding to rHb(αH87G), but it did partially occur with O2-binding to rHb(ßH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O2-binding, but its absence in the ß subunit simply enhances the O2-affinity of α subunit.

Involvement of propionate side chains of the heme in circular dichroism of myoglobin: experimental and theoretical analyses.

Incorporation of the heme into globin induces a prominent circular dichroism (CD) band in the Soret region. The appearance of heme optical activity is widely believed to arise from the interaction between the heme and aromatic residues of the globin. However, hemoglobin (Hb) containing the reversed heme exhibits a CD spectrum obviously different from that of native Hb, indicating that the interactions of heme side chains with globin contribute to the appearance of heme optical activity. We examined this possibility by comparing CD spectra of native myoglobin (Mb) and those of Mb reconstituted with synthetic hemes lacking vinyl and/or propionate. Replacement of 2,4-vinyl groups with methyl induced moderate changes. In contrast, replacement of 6,7-propionate groups with carboxylate resulted in complete disappearance of the positive Soret CD band. To get theoretical basis for the contributions of 6,7-side chains on the band, we investigated the CD spectra at a time-dependent density functional theory level. In the antiparallel conformation of the 6,7-side chains, the rotational strengths were calculated to be positive, on the other hand in the parallel conformation to be negative. We also found that the weak Soret CD band in 2,4-dimethyl-6,7-dicarboxyheme can be explained by canceling between different carboxyl conformers.

Circular dichroism of hemoglobin and myoglobin.

While heme alone does not exhibit circular dichroism (CD) spectrum, it exhibits a prominent positive CD band in the Soret region when incorporated into apoglobin of myoglobin (Mb) and hemoglobin (Hb). The appearance of this optical activity is widely accepted to arise from the interactions between the heme and aromatic residues of the globin. However, the reversed heme orientation in Hb was found to exhibit a CD spectrum obviously different from that of native Hb, indicating that the interactions of side chains of heme with globin also contribute to the appearance of heme optical activity. We examined this possibility by comparing CD spectra of native Mb and those of Mb reconstituted with unnatural heme lacking the vinyl and/or propionate. Replacement of vinyls at the 2,4-positions with methyls induced a 30% decrease in CD intensity of the positive Soret CD band without changes of spectral shape. In contrast, the replacement of the propionate at the 6,7-positions with carboxylic acid groups resulted in almost complete disappearance of the Soret CD band. These results seem to suggest that interactions of heme side chains, especially 2,4-vinyls and 6,7-propionates, with globin, as well as the electronic coupling of the heme bands with those of intrinsic protein chromophores, contribute to the appearance of the prominent positive Soret CD band of Mb and Hb.

Near-UV circular dichroism and UV resonance Raman spectra of tryptophan residues as a structural marker of proteins.

Near-UV circular dichroism (CD) and UV resonance Raman (UVRR) spectra of L-tryptophan (Trp), its derivatives, and indole-C3 derivatives were investigated to utilize Trp signals of proteins as a structural marker. CD spectra of Trp are classified into four types: Free L-Trp gives type II (around 270 nm, L(a) transition), while L-Trp in proteins generally yields type I (around 280-290 nm, L(b) transition) often with vibronic structures. All the indole-C3 derivatives except for L-Trp gave no CD bands for L(a) and L(b) transitions, indicating that the asymmetric carbon (Cα) connected through C3-Cß is essential to appearance of CD. We demonstrate here that the type of CD spectra is determined by a condition of the amino group of Trp; it was changed from type II to type I by the modification of the amino group. In contrast, the modification of the carboxyl group of L-Trp had little effects on a CD spectrum. The 229 nm excited UVRR spectra were almost the same between L-Trp and indole-C3 derivatives. Comparison of CD and UVRR spectra of Trp residues in proteins suggested that mainly the W17 (possibly together with W16) mode contributes to the characteristic vibronic coupling of L(b) transition. Both UVRR and CD spectra of L-Trp were influenced by protonation of amino and/or carboxyl groups, but those changes were distinguished from hydrogen bonding effects at N1H of indole. It is likely that these protonations are communicated to indole through σ-bonds containing Cα and thus influence both chirality of L(a) and L(b) transitions and properties of the Bb excited state.

Near-UV circular dichroism and UV resonance Raman spectra of individual tryptophan residues in human hemoglobin and their changes upon the quaternary structure transition.

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αß dimer: at α14, ß15, and ß37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (ß15Trp→Leu), and rHb (ß37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and ß15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas ß37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of ß37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.

Differences between protein dynamics of hemoglobin upon dissociation of oxygen and carbon monoxide.

Protein dynamics of human adult hemoglobin (HbA) upon ligand photolysis of oxygen (O(2)) and carbon monoxide (CO) was investigated using time-resolved resonance Raman (TR(3)) spectroscopy. The TR(3) spectra of the both photoproducts at 1-ns delay differed from that of the equilibrium deligated form (deoxy form) in the frequencies of the iron-histidine stretching [ν(Fe-His)] and methine wagging (γ(7)) modes, and the band intensity of pyrrole stretching and substituent bending (ν(8)) modes. Spectral changes of the O(2) photoproduct in the submicrosecond region were faster than those of the CO photoproduct, indicating that the structural dynamics following the photodissociation is ligand dependent for HbA. In contrast, no ligand dependence of the dynamics was observed for myoglobin, which has a structure similar to that of the subunit of HbA. The structural dynamics and relevance to the functionality of HbA also are discussed.

A new way to understand quaternary structure changes of hemoglobin upon ligand binding on the basis of UV-resonance Raman evaluation of intersubunit interactions.

The single residue vibrational spectra of tryptophan (Trp) and tyrosine (Tyr) residues in human adult hemoglobin (HbA), which play important roles in cooperative oxygen binding, were determined for the deoxy and CO-bound forms by applying UV resonance Raman spectroscopy to various variant Hbs. It was found that Trpß37, Tyrα42, Tyrα140, and Tyrß145 at the α(1)-ß(2) subunit interface underwent transitions between two contact states (named as T and R) upon ligand binding, while Trpα14, Trpß15, and Tyrß35 displayed little changes. The corresponding spectral changes were identified only for the α(2)ß(2) tetramer, but not the isolated α and ß chains in the oligomeric forms, and therefore were exclusively attributed to a quaternary structure change. Ligand binding as well as allosteric effectors and pH altered only the number of the T-contacted Tyr and Trp residues without varying the two contact states themselves. A new method to semiquantitatively evaluate the amount of T-contacted Tyr and Trp residues in a given liganded form is here proposed, and with it a quaternary structure was determined for various symmetrically half-liganded forms obtained with ligand-hybrid, metal-hybrid, and valency-hybrid Hbs. It was found that ligand binding to the α or ß subunits yielded different subunit contacts and that the contact changes of the Trp and Tyr residues were not always concerted. The contact changes at the α(1)-ß(2) (α(2)-ß(1)) interface are correlated with the proximal strain exerted on the Fe-His(F8) bond, which is noted to be much larger in the α than ß subunits in the α(2)ß(2) tetramer.

Dimer-tetramer association equilibria of human adult hemoglobin and its mutants as observed by analytical ultracentrifugation.

Dimer-tetramer equilibrium of human adult hemoglobin in CO form (COHb A) and its mutants were measured by sedimentation velocity and sedimentation equilibrium. In sedimentation velocity, the association constants were estimated by measuring the concentration dependence of the weight average sedimentation coefficients at pH 6 and 7 and fitting the data to the theoretical binding isotherms with association constants as a parameter. Association constants of wild type Hb A and three mutant Hbs, Hb Hirose(ßW37S), recombinant (r)Hb(ßW37H) and rHb(αY42S), in which an amino acid was replaced at the α(1)ß(2) interface, were measured in the presence and absence of inositol hexaphosphate (IHP). All the three mutations lowered the value of association constants, but the presence of IHP shifted the equilibrium toward tetramer. Although the association constant between dimer and tetramer of rHb(ßW37H) and rHb(αY42S) were similar, sedimentation coefficient distribution function, c(s), analysis indicated that the association and dissociation rate constants of the former is higher than the latter.

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.

Effect of reversed heme orientation on circular dichroism and cooperative oxygen binding of human adult hemoglobin.

We found that recombinant human adult hemoglobin (rHb A) expressed in Escherichia coli showed heterogeneity of components with the intensity of a positive CD band at 260 nm and that it could be resolved into three components (SP-1, SP-2, and SP-3) by SP-Sepharose column chromatography. 1H NMR revealed that SP-1 is identical with native Hb A, while SP-2 and SP-3 largely contain the reversed heme isomer in both the alpha and beta subunits, with contents of approximately 50 and >80% in SP-2 and SP-3, respectively. Rotation of the heme 180 degrees about the 5,15-meso axis (reversed heme) causes an interexchange of the methyl groups at positions 2 and 7 with the vinyl groups at positions 8 and 3, respectively. To examine the effect of the modification of the heme-protein contact on the structure and function of Hb A, we compared the 1H NMR, CD, and oxygen binding properties of the three components with those of native Hb A. Native Hb A exhibits a distinct positive CD band in both the near-UV and Soret regions, but rHb A with reversed heme exhibits a very weak positive CD band at 260 nm and a prominent negative CD band in the Soret region. Cooperativity, as measured by Hill's n value, decreased from 3.18 (SP-1) to 2.94 (SP-2) to 2.63 (SP-3) with an increase in the reversed heme orientation. The effect of an allosteric effector, inositol hexaphosphate (IHP), on the oxygen binding properties was also reduced in rHb A with reversed heme. These results indicate that changes in the heme-globin contact exert a discernible influence on CD spectra and cooperative oxygen binding.

Oxygenation properties of extracellular giant hemoglobin from Oligobrachia mashikoi.

Oxygenation properties of hemoglobin (Hb) from Oligobrachia mashikoi were extensively investigated. Compared to human Hb, Oligobrachia Hb showed a high oxygen affinity (P(50)=1.4 mmHg), low cooperativity (n =1.4), and a small Bohr effect (deltaH(+)=-0.28) at pH 7.4 in the presence of minimum salts. Addition of NaCl caused no change in the oxygenation properties of Oligobrachia Hb, indicating that Na(+) and Cl(-) had no effect. Mg(2+) and Ca(2+) remarkably increased the oxygen affinity and cooperativity. The dependence of the oxygen affinity on Ca(2+) concentration indicated that ca. 0.6 Ca(2+) per heme is bound to the protein moiety upon oxygen binding. CO(2) and a polyanion, inositol hexaphosphate, showed a null effect on the oxygenation properties. Thus, unlike the vertebrate Hbs, but like the annelid extracellular Hbs, the oxygen binding properties of Oligobrachia Hb are regulated by divalent cations which preferentially bind to the oxy form.

Quaternary structures of intermediately ligated human hemoglobin a and influences from strong allosteric effectors: resonance Raman investigation.

The Fe-histidine stretching (nu(Fe-His)) frequency was determined for deoxy subunits of intermediately ligated human hemoglobin A in equilibrium and CO-photodissociated picosecond transient species in the presence and absence of strong allosteric effectors like inositol(hexakis)phosphate, bezafibrate, and 2,3-bisphosphoglycerate. The nu(Fe-His) frequency of deoxyHb A was unaltered by the effectors. The T-to-R transition occurred around m = 2-3 in the absence of effectors but m > 3.5 in their presence, where m is the average number of ligands bound to Hb and was determined from the intensity of the nu(4) band measured in the same experiment. The alpha1-beta2 subunit contacts revealed by ultraviolet resonance Raman spectra, which were distinctly different between the T and R states, remained unchanged by the effectors. This observation would solve the recent discrepancy that the strong effectors remove the cooperativity of oxygen binding in the low-affinity limit, whereas the (1)H NMR spectrum of fully ligated form exhibits the pattern of the R state.