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.

New mechanistic insight into intramolecular arene hydroxylation initiated by (µ-1,2-peroxo)diiron(III) complexes with dinucleating ligands.

(µ-1,2-Peroxo)diiron(iii) complexes (-R) with dinucleating ligands (R-L) generated from the reaction of bis(µ-hydroxo)diiron(ii) complexes [Fe2(R-L)(OH)2](2+) (-R) with dioxygen in acetone at -20 °C provide a diiron-centred electrophilic oxidant, presumably diiron(iv)-oxo species, which is involved in aromatic ligand hydroxylation.

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.

Structure and properties of the catalytic site of nitric oxide reductase at ambient temperature.

Nitric oxide reductase (Nor) is the third of the four enzymes of bacterial denitrification responsible for the catalytic formation of laughing gas (N2O). Here we report the detection of the hyponitrite (HO-N=N-O(-)) species (νN-N=1332cm(-1)) in the heme b3 Fe-FeB dinuclear center of Nor from Paracoccus denitrificans. We have also applied density functional theory (DFT) to characterize the bimetallic-bridging hyponitrite species in the reduction of NO to N2O by Nor and compare the present results with those recently reported for the N-N bond formation in the ba3 and caa3 oxidoreductases from Thermus thermophilus.

Nitric oxide activation by caa3 oxidoreductase from Thermus thermophilus.

Visible and UV-resonance Raman spectroscopy was employed to investigate the reaction of NO with cytochrome caa3 from Thermus thermophilus. We show the formation of the hyponitrite (HO-N=N-O)(-) bound to the heme a3 species (νN=N = 1330 cm(-1)) forming a high spin complex in the oxidized heme a3 Fe/CuB binuclear center of caa3-oxidoreductase. In the absence of heme a3 Fe(2+)-NO formation, the electron required for the formation of the N=N bond originates from the autoreduction of CuB by NO, producing nitrite. With the identification of the hyponitrite intermediate the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification is fully supported and the mechanism for the 2e(-)/2H(+) reduction of NO to N2O can be described with more certainty.

Infrared and Raman spectroscopic investigation of the reaction mechanism of cytochrome c oxidase.

Recent progress in studies on the proton-pumping and O2reduction mechanisms of cytochrome c oxidase (CcO) elucidated by infrared (IR) and resonance Raman (rR) spectroscopy, is reviewed. CcO is the terminal enzyme of the respiratory chain and its O2reduction reaction is coupled with H⁺ pumping activity across the inner mitochondrial membrane. The former is catalyzed by heme a3 and its mechanism has been determined using a rR technique, while the latter used the protein moiety and has been investigated with an IR technique. The number of H⁺ relative to e⁻ transferred in the reaction is 1:1, and their coupling is presumably performed by heme a and nearby residues. To perform this function, different parts of the protein need to cooperate with each other spontaneously and sequentially. It is the purpose of this article to describe the structural details on the coupling on the basis of the vibrational spectra of certain specified residues and chromophores involved in the reaction. Recent developments in time-resolved IR and Raman technology concomitant with protein manipulation methods have yielded profound insights into such structural changes. In particular, the new IR techniques that yielded the breakthrough are reviewed and assessed in detail. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

Resonance Raman spectroscopy.

Flavin is a general name given to molecules having the heteroaromatic ring system of 7,8-dimethylisoalloxazine but practically means riboflavin (Rfl), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) in biological systems, whose structures are illustrated in Fig. 1, together with the atomic numbering scheme and ring numbering of the isoalloxazine moiety. As the isoalloxazine skeleton cannot be synthesized in human cells, it is obtained from diet as Rfl (vitamin B2). FAD and FMN can act as cofactors in flavoenzymes but Rfl does not. Most flavoenzymes catalyze redox reactions of substrates (Miura, Chem Rec 1:183-194, 2001). When O2 serves as the oxidant in the oxidation half cycle of an enzymic reaction, the enzyme is called "flavo-oxidase" but when others do, the enzyme is called "flavo-dehydrogenase." The difference between the two types of oxidative catalysis arises from delicate differences in the π-electron distributions in the isoalloxazine ring, which can be revealed by Raman spectroscopy (Miura, Chem Rec 1:183-194, 2001). Since a flavin is an extremely versatile molecule, the scientific field including chemistry, biochemistry, and enzymology is collectively called "flavonology." It was found recently, however, that the flavin also acts as a chromophore to initiate light-induced DNA repair and signal transductions (Sancar, Chem Rev 103:2203-2237, 2003).

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.

The protein effect in the structure of two ferryl-oxo intermediates at the same oxidation level in the heme copper binuclear center of cytochrome c oxidase.

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O2 activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O2 with the mixed valence form (CuA(2+), heme a(3+), heme a3(2+)-CuB(1+)) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm(-1) at the peroxy oxidation level (PM). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the PM intermediate is also formed in the reaction of Y167F with O2. These results suggest that in the fully reduced enzyme, the proton pumping ν(Fe(IV)=O) = 804 cm(-1) to ν(Fe(IV)=O) = 790 cm(-1) transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a3 but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a3. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a3-Asp-399-H2O site and, thus, to the exit/output proton channel (H2O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a3 controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.

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.

Site-specific protein dynamics in communication pathway from sensor to signaling domain of oxygen sensor protein, HemAT-Bs: Time-resolved Ultraviolet Resonance Raman Study.

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ∼50 µs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ∼50 µs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 µs exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.

Effects of the bHLH domain on axial coordination of heme in the PAS-A domain of neuronal PAS domain protein 2 (NPAS2): conversion from His119/Cys170 coordination to His119/His171 coordination.

Neuronal PAS domain protein 2 (NPAS2), which is a CO-dependent transcription factor, consists of a basic helix-loop-helix domain (bHLH), and two heme-containing PAS domains (PAS-A and PAS-B). In our previous study on the isolated PAS-A domain, we concluded that His119 and Cys170 are the axial ligands of the ferric heme, while Cys170 is replaced by His171 upon reduction of heme (Uchida et al., J. Biol. Chem. 270, (2005) 21358-21368.). Recently, we characterized the PAS-A domain combined with the N-terminal bHLH domain, and found that some spectroscopic features were different from those of the isolated PAS-A domain (Mukaiyama et al., FEBS J. 273, (2006) 2528-2539.). Therefore, we reinvestigated the coordination structure of heme in the bHLH-PAS-A domain and prepared four histidine and one cysteine mutants. Resonance Raman spectrum of the Cys170Ala mutant is the same as that of wild type with a dominant 6-coordinate heme in the ferric form. In contrast, His119Ala and His171Ala mutants significantly increase amounts of the 5-coordinate species, indicating that His119 and His171, not Cys170, are axial ligands of the ferric heme in the bHLH-PAS-A domain. We had confirmed that the coordination structure of the isolated PAS-A domain is in equilibrium between Cys-Fe-His and His-Fe-His coordinated species but newly found that interaction of the PAS-A domain with the bHLH domain shifts the equilibrium toward the latter structure. Such flexibility in the heme coordination structure seems to be in favor of signal transduction in NPAS2.

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.