Portal de Periódicos da CAPES

Modification of α-Chain or β-Chain Heme Pocket Polarity by Val(E11) → Thr Substitution Has Different Effects on the Steric, Dynamic, and Functional Properties of Human Recombinant Hemoglobin

1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês

10.1074/jbc.272.42.26271

1083-351X

Antonio Cupane, Maurizio Leone, Valeria Militello, Fred K. Friedman, Aditya P. Koley, Gregory B. Vásquez, William S. Brinigar, Michael Karavitis, Clara Fronticelli,

Mass Spectrometry Techniques and Applications

The dynamic and functional properties of mutant deoxyhemoglobins in which either the β-globin Val67(E11) or the α-globin Val62(E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified α chain and β chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the α chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the β chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified α chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the α chain distal heme pocket can bridge by H bonding between Thr62(E11) and His58(E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified β chains is not observed by x-ray crystallography. The dynamic and functional properties of mutant deoxyhemoglobins in which either the β-globin Val67(E11) or the α-globin Val62(E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified α chain and β chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the α chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the β chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified α chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the α chain distal heme pocket can bridge by H bonding between Thr62(E11) and His58(E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified β chains is not observed by x-ray crystallography. The mechanism of hemoglobin cooperativity requires the concerted action of the constituent α and β subunits. The presence of conformational differences between the α and β heme pockets is well recognized (1Fermi G. Perutz M.F. Shaanan B. Fourme R. J. Mol. Biol. 1984; 174: 159-174Crossref Scopus (673) Google Scholar, 2Shaanan B. J. Mol. Biol. 1983; 171: 31-59Crossref PubMed Scopus (524) Google Scholar, 3Fronticelli C. Pechik I. Brinigar W.S. Kowalczyk J. Gilliland G.L. J. Biol. Chem. 1994; 269: 23965-23969Abstract Full Text PDF PubMed Google Scholar). This is reflected in different functional characteristics of the α and β subunits, as higher oxygen affinity of the α subunits with respect to β subunits (4Rivetti C. Mozzarelli A. Rossi G.L. Henry E.R. Eaton W.A. Biochemistry. 1993; 32: 2888-2906Crossref PubMed Scopus (120) Google Scholar), and increased affinity of the α heme pocket for the heme (5Benesch R.E. Kwong S. J. Biol. Chem. 1990; 265: 14881-14885Abstract Full Text PDF PubMed Google Scholar). An approach to a better understanding of the factors regulating the functional properties of the heme pockets is to investigate the effect of the same mutation on the dynamics and ligand accessibility of the α and β pockets of mutant hemoglobins carrying the same amino acid substitution and to relate these effects with the conformational properties of the heme pockets. The latter can be characterized by techniques such as crystallographic analysis or molecular modeling. This approach is now feasible owing to the recent development of recombinant techniques for the expression and production of mutant hemoglobins (6Nagai K. Thogersen H.C. Nature. 1984; 309: 810-813Crossref PubMed Scopus (327) Google Scholar, 7Fronticelli C. O'Donnell J.K. Brinigar W.S. J. Protein Chem. 1991; 10: 495-501Crossref PubMed Scopus (31) Google Scholar, 8Sanna M.T. Razynska A. Karavitis M. Koley A.P. Friedman F.K. Russu I.M. Brinigar W.S. Fronticelli C. J. Biol. Chem. 1997; 272: 3478-3486Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Access of ligand to the heme group is hindered by the presence of the polypeptide chain which buries the heme in a hydrophobic crevice in the protein interior. Iron-ligand combination must therefore be accompanied by conformational fluctuations in the protein which create a pathway for ligand entry. It has been proposed that such a pathway involves concerted movements of the side chains of His(E7) and Val(E11) (9Case D.A. Karplus M. J. Mol. Biol. 1979; 132: 353-358Crossref Scopus (390) Google Scholar, 10Kottalam J. Case D.A. J. Am. Chem. Soc. 1988; 110: 7690-7697Crossref Scopus (162) Google Scholar). Several mutant hemoglobins carrying the same mutation in the α and β heme pocket have been obtained, and the association and dissociation rate constants for O2 and CO binding to R state derivatives have been measured (11Mathews A.J. Rohlfs R.J. Olson J.S. Tame J. Renaud J.-P. Nagai K. J. Biol. Chem. 1989; 264: 16573-16583Abstract Full Text PDF PubMed Google Scholar). Replacement of His(E7) with Gly and Gln affected the kinetic parameters of O2 and CO binding to the α heme but did not affect the kinetic parameters for the binding of these ligands to the β heme. Similarly, replacement of Val(E11) with Ala and Leu affected the kinetic parameters of O2 and CO binding to the α heme but not to the β heme; conversely, replacement of Val(E11) with Ile produced smaller changes in the rate of ligands binding to α than to β heme pocket mutants. These data point to a different role of His(E7) and Val(E11) in the α and β pockets of R state hemoglobin. The α and β heme pockets are hydrophobic, as the only two polar residues present are the proximal and distal histidines. In our laboratory we have extensively investigated the effect of increased polarity of the β heme pocket on a mutant hemoglobin, βV(E11)T, in which Val67(E11) was replaced by the isosteric, polar threonine (12Fronticelli C. Brinigar W.S. Olson J.S. Bucci E. Gryczynski Z. O'Donnell J.K. Kowalczyk J. Biochemistry. 1993; 32: 1235-1242Crossref PubMed Scopus (26) Google Scholar). X-ray crystallography indicated that the quaternary assembly of the molecule and the stereochemistry of the β heme pocket is not modified by the substitution. The introduction of a polar threonine at position E11 introduces the possibility of an H bond between the hydrogen atom of the threonine OH and the carbonyl backbone of His(E7) (13Pechik I. Ji X. Fidelis K. Karavitis M. Moult J. Brinigar W.S. Fronticelli C. Gilliland G.L. Biochemistry. 1996; 35: 1935-1945Crossref PubMed Scopus (17) Google Scholar). Kinetic measurements on photolyzed samples indicated that the heme pocket accessibility to CO was not modified by the mutation (12Fronticelli C. Brinigar W.S. Olson J.S. Bucci E. Gryczynski Z. O'Donnell J.K. Kowalczyk J. Biochemistry. 1993; 32: 1235-1242Crossref PubMed Scopus (26) Google Scholar). The dynamics of the carbon monoxy derivative of βV(E11)T was investigated by optical absorption spectroscopy (14Militello V. Cupane A. Leone M. Brinigar W.S. Lu A.L. Fronticelli C. Proteins Struct. Funct. Genet. 1995; 22: 12-19Crossref PubMed Scopus (6) Google Scholar), and the conformation of bound CO was studied by infrared spectroscopy (13Pechik I. Ji X. Fidelis K. Karavitis M. Moult J. Brinigar W.S. Fronticelli C. Gilliland G.L. Biochemistry. 1996; 35: 1935-1945Crossref PubMed Scopus (17) Google Scholar). The data suggested the presence of a dipolar interaction between the bound CO molecule and the hydroxyl of Thr; thus, modification of the heme pocket polarity alters the dynamics and ligand stereochemistry of the β pocket of ligand-bound hemoglobin. The recent availability of the α-globin expression system allows the introduction of the above same mutation in the heme pocket of the α subunits (8Sanna M.T. Razynska A. Karavitis M. Koley A.P. Friedman F.K. Russu I.M. Brinigar W.S. Fronticelli C. J. Biol. Chem. 1997; 272: 3478-3486Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and therefore provides the possibility of investigating the factors regulating the dynamics and functionality of the α pocket. In this paper we present comparative studies of the heme pocket deoxy derivatives of the βV(E11)T 1The abbreviations used are: βV(E11)T, human hemoglobin with a Thr at position E11 in the recombinant β chains; αV(E11)T, human hemoglobin with a Thr at position E11 in the recombinant α chains; HbA, native human hemoglobin; αrHbA, human hemoglobin with recombinant α chains; βrHbA, human hemoglobin with recombinant β chains; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. 1The abbreviations used are: βV(E11)T, human hemoglobin with a Thr at position E11 in the recombinant β chains; αV(E11)T, human hemoglobin with a Thr at position E11 in the recombinant α chains; HbA, native human hemoglobin; αrHbA, human hemoglobin with recombinant α chains; βrHbA, human hemoglobin with recombinant β chains; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. and αV(E11)T mutants using low temperature optical absorption spectroscopy, kinetics of CO rebinding, and molecular modeling. In a forthcoming paper the same parameters will be compared for the carbon monoxy derivatives. The results indicate that in deoxyhemoglobin the dynamics and heme accessibility of the heme pocket in the α chains are modified, as a result of the mutation, more than in the β heme pocket. Molecular modeling suggests that this is due to the presence in the α heme pocket of a water molecule coordinated between His58(E7) and Thr62(E11), in a manner similar to that reported for the same mutation in pig myoglobin (15Smerdon S.J. Dodson G.G. Wilkinson A.J. Gibson Q.H. Blackmore R.S. Carver T.E. Olson J.S. Biochemistry. 1991; 30: 6252-6260Crossref PubMed Scopus (87) Google Scholar). Analogous modifications were less evident in βV(E11)T, consistent with crystallographic analysis showing the absence of a water molecule in the deoxyheme pocket of this mutant hemoglobin (13Pechik I. Ji X. Fidelis K. Karavitis M. Moult J. Brinigar W.S. Fronticelli C. Gilliland G.L. Biochemistry. 1996; 35: 1935-1945Crossref PubMed Scopus (17) Google Scholar). Growth, expression, and purification of the recombinant α- and β-globins, reconstitution, and assembly into tetrameric hemoglobin followed the protocols previously described (7Fronticelli C. O'Donnell J.K. Brinigar W.S. J. Protein Chem. 1991; 10: 495-501Crossref PubMed Scopus (31) Google Scholar,8Sanna M.T. Razynska A. Karavitis M. Koley A.P. Friedman F.K. Russu I.M. Brinigar W.S. Fronticelli C. J. Biol. Chem. 1997; 272: 3478-3486Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). In the tetrameric hemoglobins obtained with this type of expression system one type of subunit is native and the other is recombinant (αrHbA and βrHbA). This allows the unambiguous assignment of observed differences to the recombinant subunit. The expression and purification of the βV(E11)T mutant has been previously described (12Fronticelli C. Brinigar W.S. Olson J.S. Bucci E. Gryczynski Z. O'Donnell J.K. Kowalczyk J. Biochemistry. 1993; 32: 1235-1242Crossref PubMed Scopus (26) Google Scholar). In the α-globin the Thr62(E11) was introduced using the Promega pAlter Mutagenesys System. The mutagenic oligonucleotide employed changed the Val codon GTC to the Thr codon ACC. The Ala63(E12) codon (GCC) was also changed to GCT which created a MspA1I site to facilitate screening for the mutation. Stocks of recombinant hemoglobins were stored in the CO form at −80 °C or in liquid nitrogen. The proteins were thawed immediately before use and diluted with the appropriate buffer. Portions of the stock solutions, thawed immediately before use and suitably diluted, were converted to the oxy form by photolysis under a flux of oxygen. Oxygen was then removed by flushing with nitrogen, and the sample was reduced by adding sodium dithionite. All steps were performed in the cold and, when dithionite was present, under anaerobic conditions. The final samples for spectrophotometric measurements were 65% (v/v) glycerol/water solutions containing 0.1 m potassium phosphate buffer (pH 7 in water, at room temperature) and ∼3·10−4m sodium dithionite; the final protein concentration was ∼10−5m in heme. Optical spectra in the spectral region 500 to 370 nm and in the temperature range 300 to 20 K were recorded in digital form at 0.4-nm intervals using a Cary 2300 spectrophotometer set at 0.4-nm bandwidth, 1-s time constant, and 40 nm/min scan speed; under these conditions the spectral resolution is about 20 cm−1 at 450 nm. The base line (cuvette + solvent) was measured at room temperature and subtracted from each spectrum (in this spectral range, the base line does not depend on temperature, and absorption due to dithionite is negligible); moreover, we stress that our samples remained homogeneous and transparent at all temperatures. All other experimental details were as described previously (16Cordone L. Cupane A. Leone M. Vitrano E. Biophys. Chem. 1986; 24: 259-275Crossref PubMed Scopus (81) Google Scholar, 17Di Pace A. Cupane A. Leone M. Vitrano E. Cordone L. Biophys. J. 1992; 63: 475-484Abstract Full Text PDF PubMed Scopus (69) Google Scholar). The spectral deconvolution used in this paper yields information on the different contributions to the line width and on the various parameters that characterize the vibrational coupling. An analytical expression for the Soret band profile at various temperatures is obtained by considering a single electronic transition coupled to Franck-Condon active vibrational modes within the adiabatic and harmonic approximation. Details on the theoretical approach used have been given in previous publications (17Di Pace A. Cupane A. Leone M. Vitrano E. Cordone L. Biophys. J. 1992; 63: 475-484Abstract Full Text PDF PubMed Scopus (69) Google Scholar,18Cupane A. Leone M. Vitrano E. Cordone L. Eur. Biophys. J. 1995; 23: 385-398Crossref PubMed Scopus (55) Google Scholar), where it has been shown that the absorbance at frequency ν can be written as a progression of Voigtians (i.e. Gaussian convolutions of Lorentzians) (Equation 1), A(ν)=Mν∑(mi)∞∏i=1NSimie−Simi!×Γν−ν0−∑i=1Nhmiνi2+Γ2⊗1ς(T),Equation 1 where M is a constant proportional to the square of the electric dipole moment, and Γ is a damping factor related to the finite lifetime of the excited state (homogeneous broadening); the product extends to all high frequency vibrational modes (i.e. with hν i ≫k B T) coupled to the electronic transition, the summations to their occupation numbers, while ν i and S i are, respectively, the frequency and the linear coupling constant for the ith high frequency mode; ν0 is the frequency of the fundamental. In Equation1 quadratic coupling of the high frequency vibrational modes is neglected. The symbol ⊗ indicates the convolution operator. Coupling of the electronic transition with low frequency modes (i.e.with modes having frequency smaller than the observed bandwidth) is treated within the "short times approximation" (19Chan C.K. Page J.B. J. Chem. Phys. 1983; 79: 5234-5250Crossref Scopus (121) Google Scholar); this brings about the convolution with a gaussian line shape and contributes the temperature-dependent terms ς2(T) and ν0(T) to the line width and peak position of the band, respectively. Further contributions to the spectral line width are inhomogeneous broadening arising from different conformational substates and heme environments (20Frauenfelder H. Parak F. Young R.D. Annu. Rev. Biophys. Biophys. Chem. 1988; 17: 451-479Crossref PubMed Scopus (882) Google Scholar). For the deoxy derivatives, it has been suggested (18Cupane A. Leone M. Vitrano E. Cordone L. Eur. Biophys. J. 1995; 23: 385-398Crossref PubMed Scopus (55) Google Scholar, 21Srajer V. Schomacker K.T. Champion P.M. Phys. Rev. Lett. 1986; 57: 6656-6670Crossref Scopus (95) Google Scholar, 22Srajer V. Champion P.M. Biochemistry. 1991; 30: 7390-7402Crossref PubMed Scopus (105) Google Scholar, 23Cupane A. Leone M. Vitrano E. Eur. Biophys. J. 1993; 21: 385-391Crossref PubMed Scopus (27) Google Scholar) that the energy of the π → π* electronic transition responsible for the Soret band depends upon the position of the iron atom relative to the heme plane (see Equation 2), and therefore, ν00=ν00(Q=0)+b Q2,Equation 2 where Q is a generalized iron coordinate representing both the out-of-plane displacement and other angular coordinates such as the histidine tilt and the azimuthal orientation of the vector connecting the iron with the histidine nitrogen, and b is a proportionality constant that reflects the electronic properties of the iron-porphyrin system. The coordinate Q is assumed to have a statistical distribution of width δ around the mean coordinate position Q0 (see Equation 3). P(Q)∝exp−[(Q−Q0)2/2δ2]Equation 3 Substitution of Equation 3 into Equation 2 yields a non-gaussian distribution of transition frequencies. The analytical expression that describes the Soret band line shape for the deoxy derivatives (18Cupane A. Leone M. Vitrano E. Cordone L. Eur. Biophys. J. 1995; 23: 385-398Crossref PubMed Scopus (55) Google Scholar, 21Srajer V. Schomacker K.T. Champion P.M. Phys. Rev. Lett. 1986; 57: 6656-6670Crossref Scopus (95) Google Scholar,23Cupane A. Leone M. Vitrano E. Eur. Biophys. J. 1993; 21: 385-391Crossref PubMed Scopus (27) Google Scholar) is shown in Equation 4. A(ν)=V(ν)⊗12δ(ν−ν0)2πbe−(ν−ν0)1/2+Q0b22bδ2+e−(ν−ν0)1/2−Q0b22bδ2Equation 4 V(ν) is given by Equation 1. The first step of the data analysis is based on fittings of the experimental Soret absorption spectra with Equation 4. The analysis is performed on a Microway I860 add-on board for IBM-PC, using a Marquardt-type nonlinear least squares algorithm (24Marquardt D.W. J. Soc. Ind. Appl. Math. 1963; 11: 431-441Crossref Google Scholar). The fitting parameters are M, Γ, S i, ς, ν o, Q0 b, and δ b; the values relative to high frequency modes are taken from resonance Raman spectra of human deoxyhemoglobin reported in the literature (25Bangcharoenpaurpong O. Schomacker K.T. Champion P.M. J. Am. Chem. Soc. 1984; 106: 5688-5698Crossref Scopus (134) Google Scholar). As in previous work (14Militello V. Cupane A. Leone M. Brinigar W.S. Lu A.L. Fronticelli C. Proteins Struct. Funct. Genet. 1995; 22: 12-19Crossref PubMed Scopus (6) Google Scholar), we assume that the mutations investigated do not alter the frequency of these modes; this assumption is supported by the excellent quality of the fittings obtained (see Fig. 2 and Table I). The most coupled modes are those centered at 370, 674, and 1357 cm−1, whereas other less coupled modes do not contribute significantly to the line shapes and are therefore neglected. It is worth mentioning that in the resonance Raman spectra of hemoglobin the frequencies 674 cm−1 and 1357 cm−1 correspond to very sharp lines and are thought to arise from in-plane vibrational modes of the heme group (the well known ν7 and ν4respectively; Ref. 26Spiro T.G. Lever A.B.P. Gray H.B. Iron Porphyrins. Addison-Wesley Publishing Co., Reading, MA1983: 89-159Google Scholar). On the contrary, 370 cm−1 is the "average effective" frequency accounting for a spectral region characterized by several quasi-degenerate peaks; both in-plane and out-of-plane modes contribute to these spectral regions.S i parameters are fixed at the low temperature values determined from the clearly resolved vibronic structure of the band; in this way we avoid fitting ambiguities arising from broadening of the band and consequent lack of a clearly resolved vibronic structure at high temperature.Table ILow temperature (T = 20 K) linear coupling constants of the high frequency modes and Γ values for the deoxy derivatives of the mutant hemoglobins investigatedS 370S 674S L357ΓQs bδ bcm −1HbA1-aData relative to HbA and to SwMb have been taken from Ref. 23.0.11 ± 0.020.21 ± 0.020.05 ± 0.01175 ± 90.18 ± 0.030.20 ± 0.03βV(E11)T0.04 ± 0.020.18 ± 0.030.04 ± 0.02184 ± 100.22 ± 0.030.19 ± 0.03βrHbA0.09 ± 0.020.16 ± 0.030.06 ± 0.02190 ± 100.19 ± 0.030.19 ± 0.03αV(E11)T0.05 ± 0.020.21 ± 0.020.06 ± 0.02190 ± 100.19 ± 0.030.14 ± 0.03αrHbA0.05 ± 0.020.27 ± 0.030.03 ± 0.01186 ± 100.19 ± 0.030.20 ± 0.03SwMb1-aData relative to HbA and to SwMb have been taken from Ref. 23.0.32 ± 0.020.24 ± 0.020.09 ± 0.01180 ± 100.17 ± 0.010.16 ± 0.021-a Data relative to HbA and to SwMb have been taken from Ref. 23Cupane A. Leone M. Vitrano E. Eur. Biophys. J. 1993; 21: 385-391Crossref PubMed Scopus (27) Google Scholar. Open table in a new tab The second step includes the temperature dependence of parameter ς2 (i.e. the gaussian width of the band) in Equation 4. This is done within the Einstein harmonic oscillator approximation and considering the coupling of the Soret band with a "bath" of low frequency modes (18Cupane A. Leone M. Vitrano E. Cordone L. Eur. Biophys. J. 1995; 23: 385-398Crossref PubMed Scopus (55) Google Scholar); within this model (Equation 5) one has ς2=NS 2coth(h〈ν〉/2kT),Equation 5 where and S are the effective frequency and linear coupling constants of the low frequency bath, and Nis the number of soft modes. The ς2 increase with temperature predicted by Equation 5 simply reflects the well known amplitude increase of harmonic nuclear motions as the temperature is increased. As mentioned above, the effect of inhomogeneous broadening is taken into account by the parameter δ b. The ς2 thermal behavior therefore gives information on dynamic properties (linear coupling with the low frequency bath of the system) that involve motions not only of the chromophore but also of the heme pocket and of larger parts of the protein. Complementary information on the stereodynamic properties of the heme pocket is also obtained from the parameter ν0(i.e. the peak frequency of the band); in fact, its temperature dependence reflects not only the quadratic coupling of the electronic transition with the bath of soft modes but also the local electric field experienced by the chromophore within its surroundings. Flash photolysis was carried out in solutions containing 5 μm heme and 50 μm CO at 23 °C in 0.1 m Bis-Tris (pH 7.0) containing 0.1m KCl. Approximately 0.5 mg of sodium dithionite was added to reduce any ferric heme to the ferrous state. The instrumentation and experimental details for laser flash photolysis were essentially as described previously (27Markowitz A. Robinson R.C. Omata Y. Friedman F.K. Anal. Instrum. 1992; 20: 213-221Crossref Scopus (0) Google Scholar). A pulse (0.6 μs) from a dye laser disrupted the photolabile heme Fe–CO bond, and the recombination of CO with the hemeprotein was then monitored by following the absorbance change at 436 nm. Data were transmitted to a microcomputer for processing and analysis. Standard multiexponential analysis of the kinetic data was performed according to Equation 6. ΔA(t)=∑i=1naiexp(−kit)Equation 6 ΔA(t) is the total absorbance change observed at time t, a i is the absorbance change for component i at t = 0,k i is the observed pseudo-first order rate constant for component i, and n is the number of independent components. Least squares analysis was performed with RS/1 software (BBN software products, Cambridge, MA) on a Dell 450/ME microcomputer. Statistical significance (p < 0.05) was evaluated using the Student's t test, assuming equal variances for all the experimental points. Molecular modeling was carried out with the program O (28Jones T.A. Zou J.-V. Cowan S.W. Kjeldgaard M. Acta Crystallogr. 1991; A47: 110-119Crossref Scopus (13006) Google Scholar). Comparison of the heme pockets of deoxymyoglobin with a Thr at position E11 (molecule B of the structure 1YCB) and of deoxyhemoglobin (molecule α2 of the structure 2HHD) was done using the program ALIGN (29Satow Y. Cohen G. Padlan E.A. Davies D.R. J. Mol. Biol. 1986; 190: 593-604Crossref PubMed Scopus (532) Google Scholar). Fig. 1shows the spectra of deoxy-βV(E11)T at various temperatures between 300 and 20 K. As can be seen, the band is markedly asymmetric at all temperatures; this asymmetry is accounted for by the asymmetric distribution of transition frequencies in Equation 4. The bandwidth of the Soret band decreases as the temperature is lowered, and the asymmetry is almost constant; moreover, a slight blue shift of the band position upon lowering the temperature is also present. Fig.2 shows the deconvolution of the 70 K spectrum of αV(E11)T in terms of Equation 4, together with the residuals on an expanded scale. A fitting of analogous quality is obtained also for βV(E11)T; for both proteins the fitting quality improves at higher temperatures. The values of the linear coupling constants for the high frequency modes (S i ) and of the Lorentzian width (Γ) are listed in TableI, in comparison with the values obtained for the recombinant wild type proteins αrHbA and βrHbA, for native human hemoglobin (HbA), and for sperm whale myoglobin (SwMb). As can be seen, no relevant effect on these parameters is observed; this indicates that the mutations, both in the α and in the β chain, do not affect the local electronic and vibrational properties of the chromophore. In particular, parameters Q0 band δ b are unaffected, thus suggesting that the iron-heme-proximal histidine geometry is also unaffected. The temperature dependence of parameter ν0 for the mutants αV(E11)T and βV(E11)T is reported in the left panels of Fig. 3, together with the analogous quantities relative to the recombinant wild type proteins. Values relative to HbA and to SwMb are reported in the right panels of the same figure, for the sake of comparison. From Fig. 3it can be seen that both the HbA recombinant proteins exhibit the same ν0 behavior as HbA; for the mutant βV(E11)T there is a very small effect while, for αV(E11)T, the ν0 behavior is markedly different and rather similar to that exhibited by SwMb. The difference is clearly evident at low temperatures, where a blue shift of ν0 values upon lowering the temperature is observed for HbA, αrHbA, βrHbA, and βV(E11)T, while SwMb is characterized by a red shift; ν0 values relative to αV(E11)T have an intermediate behavior and remain approximately constant. The temperature dependence of parameter ς2 for the investigated proteins is reported in Fig.4. As can be seen, both mutants exhibit a sharper ς2 temperature dependence with respect to the wild types and to HbA; moreover, the α chain mutant has lower ς2 values at low temperatures. The continuous lines represent the fittings of the data in the temperature range 20–120 K in terms of Equation 5; values of the parametersNS and obtained from the fittings are reported in Table II. The two mutants are characterized by lower values of the average frequency of the soft modes, and the α chain mutant exhibits also a lower value of parameter NS. At temperatures higher than 120 K an increase of ς2 values well above the predictions of Equation 5 is observed. Since Equation 5 has been derived within the harmonic approximation, we attribute these deviations to the onset of large amplitude non-harmonic nuclear motions (17Di Pace A. Cupane A. Leone M. Vitrano E. Cordone L. Biophys. J. 1992; 63: 475-484Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 18Cupane A. Leone M. Vitrano E. Cordone L. Eur. Biophys. J. 1995; 23: 385-398Crossref PubMed Scopus (55) Google Scholar). An increase of average atomic fluctuations well above the prediction of the harmonic behavior and occurring at high temperatures has been observed, for hemeproteins, with a variety of experimental techniques (30Doster W. Cusack S. Petry W. Nature. 1989; 337: 754-756Crossref PubMed Scopus (978) Google Scholar, 31Parak F. Knapp E.W. Kucheida W. J. Mol. Biol. 1982; 161: 177-194Crossref PubMed Scopus (289) Google Scholar, 32Loncharich R.J. Brooks B.R. J. Mol. Biol. 1990; 215: 439-455Crossref PubMed Scopus (124) Google Scholar, 33Melchers B. Knapp E.W. Parak F. Cordone L. Cupane A. Leone M. Biophys. J. 1996; 70: 2092-2099Abstract Full Text PDF PubMed Scopus (78) Google Scholar) and has been attributed to a transition in protein mobility from the low temperature "solid like" behavior characterized by essentially harmonic oscillations of the nuclei around their equilibrium positions, to a high temperature "liquid like" behavior characterized by the jumping among different conformational substates of the biomolecule. It should be noted that, for deoxyhemoglobin and myoglobin, the onset of anharmonic motions occurs at about 120 K, i.e. well before the temperature at which the glass transition of the glycerol/water solvent mixture occurs (∼180 K) (34Cordone L. Cupane A. Leone M. Vitrano E. Bulone D. J. Mol. Biol. 1988; 199: 213-218Crossref PubMed Scopus (22) Google Scholar, 35Ansari A. Berendzen J. Braunstein D. Cowen B.R. Frauenfelder H. Hong M.K. Iben I.E.T. Johnson J.B. Ormos P. Sauke T.B. Scholl R. Schulte A. Steinbach P.J. Vittitow J. Young R.D. Biophys. Chem. 1987; 26: 337-355Crossref PubMed Scopus (340) Google Scholar, 36Leone M. Cupane A. Militello V. Cordone L. Eur. Biophys. J. 1994; 23: 349-352Crossref PubMed Scopus (28) Google Scholar).