Structural Dynamics of Myoglobin
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m109892200
ISSN1083-351X
AutoresDon C. Lamb, Karin Nienhaus, Alessandro Arcovito, Federica Draghi, A.E. Miele, Maurizio Brunori, G. Ulrich Nienhaus,
Tópico(s)Porphyrin Metabolism and Disorders
ResumoFourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A0 and A3, which can be distinguished by their different CO bands near 1965 and 1933 cm−1. They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C′, in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C′′) has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at ∼180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C′, C′′, and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C′) is severely restricted by introduction of the bulky Phe side chain at position 107. Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A0 and A3, which can be distinguished by their different CO bands near 1965 and 1933 cm−1. They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C′, in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C′′) has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at ∼180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C′, C′′, and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C′) is severely restricted by introduction of the bulky Phe side chain at position 107. Myoglobin (Mb), 1The abbreviations used are: MbmyoglobinFTIR-TDSFourier transform infrared/temperature derivative spectroscopywtwild type 1The abbreviations used are: MbmyoglobinFTIR-TDSFourier transform infrared/temperature derivative spectroscopywtwild type the first protein for which structure was solved to atomic resolution (1.Kendrew J.C. Dickerson R.E. Strandberg B.E. Hart R.G. Davis D.D. Phillips D.C. Shore V.C. Nature. 1960; 185: 422-427Crossref PubMed Scopus (854) Google Scholar), is an α-helical polypeptide chain of about 150 amino acids encapsulating a heme group to which small gaseous ligands like O2, CO, or NO can bind. Early on it was recognized that the ligand binding site in the interior of the protein is not accessible by ligands in the average structure, and it became apparent that ligand binding has to rely on structural fluctuations that provide transient channels for the ligands to enter and exit the protein. Hence, for more than 40 years Mb has played the role of a model system for the investigation of the interrelationships among structure, dynamics, and function in heme proteins and in proteins at large (2.Antonini E. Brunori M. Neuberger A. Tatum E.L. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland, Amsterdam1971Google Scholar, 3.Perutz M.F. Mathews F.S. J. Mol. Biol. 1966; 21: 199-207Crossref PubMed Scopus (241) Google Scholar, 4.Austin R.H. Beeson K.W. Eisenstein L. Frauenfelder H. Gunsalus I.C. Biochemistry. 1975; 14: 5355-5373Crossref PubMed Scopus (1317) Google Scholar, 5.Frauenfelder H. Sligar S.G. Wolynes P.G. Science. 1991; 254: 1598-1603Crossref PubMed Scopus (2558) Google Scholar, 6.Nienhaus G.U. Young R.D. Trigg G. Encyclopedia of Applied Physics. 15. VCH Publishers, New York1996: 163-184Google Scholar, 7.Ansari A. Jones C.M. Henry E.R. Hofrichter J. Eaton W.A. Biochemistry. 1994; 33: 5128-5145Crossref PubMed Scopus (199) Google Scholar, 8.Gibson Q.H. J. Biol. Chem. 1989; 264: 20155-20158Abstract Full Text PDF PubMed Google Scholar, 9.Olson J.S. Phillips Jr., G.N. J. Biol. Chem. 1996; 271: 17593-17596Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). myoglobin Fourier transform infrared/temperature derivative spectroscopy wild type myoglobin Fourier transform infrared/temperature derivative spectroscopy wild type All ligands that bind to the ferrous heme iron can be photodissociated (10.Gibson Q.H. Ainsworth S. Nature. 1957; 180: 1416-1417Crossref PubMed Scopus (87) Google Scholar), and the ensuing ligand binding reaction can be conveniently studied using time-resolved spectroscopy. After photodissociation at room temperature, the ligand remains momentarily trapped within the protein before diffusing out into the solvent. Intramolecular rebinding to the heme iron, called geminate recombination, is controlled by ligand migration within the protein matrix and thus has been exploited to investigate the internal dynamics in a time range from ps to μs (8.Gibson Q.H. J. Biol. Chem. 1989; 264: 20155-20158Abstract Full Text PDF PubMed Google Scholar, 11.Lim M. Jackson T.A. Anfinrud P.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5801-5804Crossref PubMed Scopus (148) Google Scholar, 12.Scott E.E. Gibson Q.H. Olson J.S. J. Biol. Chem. 2001; 276: 5177-5188Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). The complexity of the events controlling geminate recombination was particularly apparent in flash photolysis experiments carried out at cryogenic temperatures, which give rise to a sequence of well resolved kinetic features (4.Austin R.H. Beeson K.W. Eisenstein L. Frauenfelder H. Gunsalus I.C. Biochemistry. 1975; 14: 5355-5373Crossref PubMed Scopus (1317) Google Scholar, 13.Steinbach P.J. Ansari A. Berendzen J. Braunstein D. Chu K. Cowen B.R. Ehrenstein D. Frauenfelder H. Johnson J.B. Lamb D.C. Luck S. Mourant J.R. Nienhaus G.U. Ormos P. Philipp R. Xie A. Young R.D. Biochemistry. 1991; 30: 3988-4001Crossref PubMed Scopus (371) Google Scholar, 14.Post F. Doster W. Karvounis G. Settles M. Biophys. J. 1993; 66: 1833-1842Abstract Full Text PDF Scopus (65) Google Scholar). Propositions as to the nature of the kinetic intermediates included structural transitions of the protein that affect the ligand binding energetics and/or migration of the ligand to different docking sites (13.Steinbach P.J. Ansari A. Berendzen J. Braunstein D. Chu K. Cowen B.R. Ehrenstein D. Frauenfelder H. Johnson J.B. Lamb D.C. Luck S. Mourant J.R. Nienhaus G.U. Ormos P. Philipp R. Xie A. Young R.D. Biochemistry. 1991; 30: 3988-4001Crossref PubMed Scopus (371) Google Scholar, 14.Post F. Doster W. Karvounis G. Settles M. Biophys. J. 1993; 66: 1833-1842Abstract Full Text PDF Scopus (65) Google Scholar, 15.Powers L. Chance B. Chance M. Campbell B. Friedman J. Khalid S. Kumar C. Naqui A. Reddy K.S. Zhou Y. Biochemistry. 1987; 26: 4785-4796Crossref PubMed Scopus (74) Google Scholar, 16.Ahmed A.M. Campbell B.F. Caruso D. Chance M.R. Chavez M.D. Courtney S.H. Friedman J.M. Iben I.E.T. Ondrias M.R. Yang M. Chem. Phys. 1991; 158: 329-351Crossref Scopus (56) Google Scholar). These models were, in the absence of direct structural information, merely speculative. Nevertheless, the kinetic and spectroscopic experiments, particularly those using temperature-derivative spectroscopy (TDS) after extended illumination (17.Mourant J.R. Braunstein D.P. Chu K. Frauenfelder H. Nienhaus G.U. Ormos P. Young R.D. Biophys. J. 1993; 65: 1496-1507Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 18.Nienhaus G.U. Mourant J.R. Chu K. Frauenfelder H. Biochemistry. 1994; 33: 13413-13430Crossref PubMed Scopus (107) Google Scholar, 19.Nienhaus G.U. Chu K.C. Jesse K. Biochemistry. 1998; 37: 6819-6823Crossref PubMed Scopus (23) Google Scholar), were invaluable to the establishment of illumination protocols that populated photoproduct states to a level amenable to structural studies using x-ray crystallography. The first photoproduct structures of MbCO were obtained at 40 K (20.Schlichting I. Berendzen J.G.N. Phillips J. Sweet R.M. Nature. 1994; 371: 808-812Crossref PubMed Scopus (338) Google Scholar, 21.Teng T. S̆rajer V. Moffat K. Nat. Struct. Biol. 1994; 1: 701-705Crossref PubMed Scopus (192) Google Scholar, 22.Hartmann H. Zinser S. Komninos P. Schneider R.T. Nienhaus G.U. Parak F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7013-7016Crossref PubMed Scopus (145) Google Scholar) revealing that, after photodissociation, the CO was located in the vicinity of the heme, in the so-called primary docking site, as proposed by IR spectroscopic studies (23.Braunstein D.P. Chu K. Egeberg K.D. Frauenfelder H. Mourant J.R. Nienhaus G.U. Ormos P. Sligar S.G. Springer B.A. Young R.D. Biophys. J. 1993; 65: 2447-2454Abstract Full Text PDF PubMed Scopus (87) Google Scholar). Subsequently, new photoproduct structures of sperm whale Mb mutants became available, showing that CO can migrate away from the primary docking site into internal cavities within the protein (24.Brunori M. Vallone B. Cutruzzolà F. Travaglini-Allocatelli C. Berendzen J. Chu K. Sweet R.M. Schlichting I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2058-2063Crossref PubMed Scopus (142) Google Scholar, 25.Ostermann A. Waschipky R. Parak F.G. Nienhaus G.U. Nature. 2000; 404: 205-208Crossref PubMed Scopus (357) Google Scholar, 26.Chu K. Vojtchovsky J. McMahon B.H. Sweet R.M. Berendzen J. Schlichting I. Nature. 2000; 403: 921-923Crossref PubMed Scopus (228) Google Scholar, 27.Schlichting I. Chu K. Curr. Opin. Struct. Biol. 2000; 10: 744-752Crossref PubMed Scopus (66) Google Scholar). These cavities have long been known because of their ability to bind an atom of xenon (28.Schoenborn B.P. Watson H.C. Kendrew J.C. Nature. 1965; 207: 28-30Crossref PubMed Scopus (179) Google Scholar, 29.Tilton R.F. Kuntz I.D. Petsko G.A. Biochemistry. 1984; 23: 2849-2857Crossref PubMed Scopus (457) Google Scholar). Using nanosecond time-resolved x-ray crystallography at room temperature, Moffat and collaborators (30.S̆rajer V. Ren Z. Teng T.-Y. Schmidt M. Ursby T. Bourgeois D. Pradervand C. Schildkamp W. Wulff M. Moffat K. Biochemistry. 2001; 40: 13802-13815Crossref PubMed Scopus (284) Google Scholar) recently confirmed that these cavities play a role as transient docking sites in the physiological ligand binding process, as suggested earlier by Elber and Karplus (31.Elber R. Karplus M. J. Am. Chem. Soc. 1990; 112: 9161-9175Crossref Scopus (476) Google Scholar). For the sake of clarity, in Fig. 1 we depict a schematic view of the heme and a few of the key amino acid side chains on the distal and proximal side, as well as the approximate CO locations in the different photoproduct states (A–D) that will be referred to throughout the paper. Photoproduct structure determination is a difficult pursuit, and it is successful only if an intermediate can be populated to a significant fraction. By contrast, IR spectroscopy is a sensitive technique that allows one to distinguish different species with fractional populations in the percent range. Moreover, measurements carried out as a function of temperature yield information on the energy barriers governing internal migration and rebinding of the photodissociated ligand; particularly revealing are IR-spectroscopic experiments monitoring the stretch bands of heme-bound or photodissociated CO near 5 μm (32.Alben J.O. Beece D. Bowne S.F. Doster W. Eisenstein L. Frauenfelder H. Good D. McDonald J.D. Marden M.C. Moh P.P. Reinisch L. Reynolds A.H. Shyamsunder E. Yue K.T. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3744-3748Crossref PubMed Scopus (141) Google Scholar,33.Shimada H. Caughey W.S. J. Biol. Chem. 1982; 257: 11893-11900Abstract Full Text PDF PubMed Google Scholar). Measurements of the ligand-bound species yield detailed information about structural properties, in particular heterogeneity, in the vicinity of the active site. Multiple lines observed in the wave number range around 1950 cm−1 have been associated with multiple conformations of the protein, called the "A" substates (see Fig. 1). For example, native sperm whale Mb exhibits three A substates (A0 at ∼1966 cm−1, A1at ∼1945 cm−1, and A3 at ∼1933 cm−1) (17.Mourant J.R. Braunstein D.P. Chu K. Frauenfelder H. Nienhaus G.U. Ormos P. Young R.D. Biophys. J. 1993; 65: 1496-1507Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 34.Ansari 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 (338) Google Scholar). Comparative studies of many distal pocket mutants have shown that multiple A substates originate from electrostatic interactions between the bound CO and different heme pocket environments (23.Braunstein D.P. Chu K. Egeberg K.D. Frauenfelder H. Mourant J.R. Nienhaus G.U. Ormos P. Sligar S.G. Springer B.A. Young R.D. Biophys. J. 1993; 65: 2447-2454Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 35.Li T. Quillin M.L.G.N. Phillips J. Olson J.S. Biochemistry. 1994; 33: 1433-1446Crossref PubMed Scopus (325) Google Scholar), which alter the electron distribution and bond order of the iron-carbonyl complex and, accordingly, shift the IR stretch frequency (36.Phillips Jr., G.N. Teodoro M.L. Li T. Smith B. Olson J.S. J. Phys. Chem. B. 1999; 103: 8817-8829Crossref Scopus (241) Google Scholar). The IR bands of CO in aqueous solutions are too broad to be detected. Photodissociated CO trapped inside the protein matrix, however, displays multiple IR lines near 2130 cm−1(32.Alben J.O. Beece D. Bowne S.F. Doster W. Eisenstein L. Frauenfelder H. Good D. McDonald J.D. Marden M.C. Moh P.P. Reinisch L. Reynolds A.H. Shyamsunder E. Yue K.T. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3744-3748Crossref PubMed Scopus (141) Google Scholar) that arise from different locations and/or orientations of the CO within the protein. Although their small absorbances and wave number shifts demand careful experiments, these IR bands are useful tools to follow CO trapped inside the protein. In this work, we address the problem of ligand migration, employing two mutants of sperm whale Mb and exploiting the power of low temperature infrared spectroscopy in combination with a temperature ramp protocol. The general purpose of our work is 2-fold, i.e. to understand the role of the internal cavities and packing defects in controlling ligand migration to the heme iron and to test the power of protein engineering in dictating internal pathways and occupation of the available docking sites (see Fig. 1). The experimental approach of TDS-FTIR spectroscopy enables us to separate different dynamic processes by their different temperature dependences and to determine the enthalpy barriers governing the observed rate processes. Using this technique we investigated two multiple mutants of sperm whale Mb that seemed particularly suitable as model systems in view of their interesting and almost unique properties. The results obtained by FTIR-TDS have been correlated with the crystallographic structure obtained for both mutants in the CO-bound state. The triple Mb mutant referred to as YQR has the substitutions L29(B10)Y, H64(E7)Q, and T67(E10)R; the quadruple mutant YQRF has an additional mutation, I107(G8)F. In these Mbs, originally designed to mimic the distal pocket structure of Ascaris suum hemoglobin (37.DeBaere I. Perutz M.F. Kiger L. Marden M.C. Poyart C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1594-1597Crossref PubMed Scopus (100) Google Scholar), the Tyr at 29(B10) assists in trapping the dioxygen at the active site by an additional hydrogen bond. For YQR very peculiar ligand binding properties were observed (38.Brunori M. Cutruzzolà F. Savino C. Travaglini-Allocatelli C. Vallone B. Gibson Q.H. Biophys. J. 1999; 76: 1259-1269Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), suggesting that the ligands might easily migrate away from the primary docking site to a secondary site. The x-ray structure of YQR obtained at 30 K (24.Brunori M. Vallone B. Cutruzzolà F. Travaglini-Allocatelli C. Berendzen J. Chu K. Sweet R.M. Schlichting I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2058-2063Crossref PubMed Scopus (142) Google Scholar) showed that, after photodissociation, CO migrates into the Xe4 cavity in the back of the distal heme pocket (see Fig. 1). Instead of Ile at 107(G8), the quadruple mutant YQRF has the bulkier Phe chain protruding into the channel leading to the Xe4 cavity, which should gate access to this docking site. In this study we present a detailed picture of the structural dynamics of both mutants at low temperatures with particular focus on the effect of the mutations in the ligand migration pathways and discuss the level of consistency with room temperature kinetic experiments and low temperature crystallographic data. Mutant sperm whale myoglobins YQR and YQRF were expressed in Escherichia coli and purified as described previously (39.Travaglini-Allocatelli C. Cutruzzolà F. Brancaccio A. Vallone B. Brunori M. FEBS Lett. 1994; 352: 63-66Crossref PubMed Scopus (37) Google Scholar). Samples were prepared by dissolving the lyophilized protein at a concentration of ∼15 mm in cryosolvent (75% glycerol, 25% potassium phosphate buffer (v/v), pH 7) and subsequent reduction with excess dithionite under a CO atmosphere. For the spectroscopic experiments, a few microliters of the protein solution were held between two CaF2 windows (diameter 25.4 mm) separated by a 75-μm thick Mylar washer. The windows were sandwiched inside a block of oxygen-free high conductivity copper mounted on the cold-finger of a closed cycle helium refrigerator (model SRDK-205AW, Sumitomo, Tokyo, Japan), which allowed adjustment of the sample temperature in the range between 3 and 320 K. The temperature was measured with a silicon temperature sensor diode and regulated by a digital temperature controller (model 330, Lake Shore Cryotronics, Westerville, OH). Samples were photolyzed with light from a continuous wave, frequency-doubled Nd-YAG laser (model Forte 530–300, Laser Quantum, Manchester, UK), emitting 300 milliwatts of output power at 532 nm. The beam was split and focused with lenses on the sample from both sides. The standard photolysis rate, kL, was determined as ∼20 s−1 at low temperatures. A Fourier transform infrared (FTIR) spectrometer (IFS 66v/S, Bruker, Karlsruhe, Germany) was used to collect transmission spectra in the mid-infrared range between 1800 and 2400 cm−1 at a resolution of 2 cm−1. To assess the kinetic properties of different photoproducts, we use TDS, an experimental protocol designed to investigate thermally activated rate processes that are characterized by distributed enthalpy barriers. The method has been described in detail in previous papers (17.Mourant J.R. Braunstein D.P. Chu K. Frauenfelder H. Nienhaus G.U. Ormos P. Young R.D. Biophys. J. 1993; 65: 1496-1507Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 18.Nienhaus G.U. Mourant J.R. Chu K. Frauenfelder H. Biochemistry. 1994; 33: 13413-13430Crossref PubMed Scopus (107) Google Scholar, 19.Nienhaus G.U. Chu K.C. Jesse K. Biochemistry. 1998; 37: 6819-6823Crossref PubMed Scopus (23) Google Scholar, 40.Berendzen J. Braunstein D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1-5Crossref PubMed Scopus (78) Google Scholar), and thus we give only a short summary here. In a TDS measurement, the sample is initially perturbed from equilibrium. Here we use laser irradiation under different illumination protocols so as to enhance particular photoproduct species (18.Nienhaus G.U. Mourant J.R. Chu K. Frauenfelder H. Biochemistry. 1994; 33: 13413-13430Crossref PubMed Scopus (107) Google Scholar). The temperature is subsequently increased linearly in time at a warming rate α = 5 mK/s, and spectra are collected continuously, yielding one complete spectrum for every kelvin temperature increase. Difference spectra are calculated from spectra at consecutive temperatures and plotted as a function of temperature in a TDS map, which is a (linearly or logarithmically scaled) contour plot of the absorbance changes on a surface spanned by the wave number and temperature axes. In the experiments presented here, absorbance changes arise from two different rate processes, CO rebinding to the heme iron and CO moving to other locations. At the lowest temperatures, thermal energy is too small to overcome most of the enthalpy barriers governing these processes. As the temperature increases with time, successively higher barriers can be surmounted. Therefore, the TDS protocol ensures that the different rate processes in the sample are sorted according to their activation enthalpy, which is, to a good approximation, linearly related to the temperature at which the process is observed (40.Berendzen J. Braunstein D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1-5Crossref PubMed Scopus (78) Google Scholar). Infrared difference spectra of the YQR and YQRF mutants MbCO (Fig. 2) show that in the region of the stretch absorption of the heme-bound CO, both mutants display two A substate bands, with slightly different frequencies in YQR and YQRF. In the same figure, the IR region of the photodissociated CO displays a number of bands between 2110 and 2160 cm−1. The two main bands are located at 2142 and 2144 cm−1; the weaker bands at lower wave numbers, however, cannot be resolved unambiguously in the difference spectrum at 3 K. In MbCO, an IR absorption line near 1965 cm−1 is present in mutants with an apolar distal pocket, for example upon replacement of the distal His-64(E7) with a small aliphatic residue (23.Braunstein D.P. Chu K. Egeberg K.D. Frauenfelder H. Mourant J.R. Nienhaus G.U. Ormos P. Sligar S.G. Springer B.A. Young R.D. Biophys. J. 1993; 65: 2447-2454Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 35.Li T. Quillin M.L.G.N. Phillips J. Olson J.S. Biochemistry. 1994; 33: 1433-1446Crossref PubMed Scopus (325) Google Scholar). In wt MbCO, the A0 line at 1966 cm−1 appears upon protonation of the distal histidine with a p K of 4.5 (41.Müller J.D. McMahon B.H. Chien E.Y.T. Sligar S.G. Nienhaus G.U. Biophys. J. 1999; 77: 1036-1051Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), which forces the imidazole side chain into an alternate conformation away from the distal heme pocket into the solvent so as to better solvate the charged imidazolium side chain. The other two A substates, A1 (1945 cm−1) and A3 (1933 cm−1), are characterized by a neutral imidazole side chain residing in the heme pocket. Johnson et al. (42.Johnson J.B. Lamb D.C. Frauenfelder H. Müller J.D. McMahon B.H. Nienhaus G.U. Young R.D. Biophys. J. 1996; 71: 1563-1573Abstract Full Text PDF PubMed Scopus (138) Google Scholar) measured the exchange kinetics between the A1 and A3 states and suggested that they originate from two slightly different conformations of His-64 in the distal pocket. Additional support for this view was provided recently on the basis of a high resolution crystal analysis (43.Vojtechovsky J. Chu K. Berendzen J. Sweet R.M. Schlichting I. Biophys. J. 1999; 77: 2153-2174Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). An enhanced A3-like absorption was noticed in several mutants modified at residue 29, for example L29A/L29V/L29I/L29F (35.Li T. Quillin M.L.G.N. Phillips J. Olson J.S. Biochemistry. 1994; 33: 1433-1446Crossref PubMed Scopus (325) Google Scholar). Presumably, interactions between Leu-29 and His-64 are responsible for the occurrence of A1 and A3 in native MbCO. Although the two mutants studied here have residues Leu-29 and His-64 replaced by Tyr and Gln, respectively, we observe an A state spectrum with bands similar to A0 and A3 of the native protein. Based on data obtained on the H64Q mutant of sperm whale MbCO (35.Li T. Quillin M.L.G.N. Phillips J. Olson J.S. Biochemistry. 1994; 33: 1433-1446Crossref PubMed Scopus (325) Google Scholar), an A1-like line may have been expected, because the conformation of the glutamine side chain places its Nε-bound hydrogen in a position similar to that of the imidazole Nε of His-64. The double mutant L29F/H64Q has a broad, further red-shifted IR absorption at 1938 cm−1(44.Zhao X. Vyas K. Nguyen B.D. Rajarathnam K. Mar G.N.L. Li T. Phillips G.N. Eich R.F. Olson J.S. Ling J. Bocian D.F. J. Biol. Chem. 1995; 270: 20763-20774Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), whereas the L29F single mutant has a fairly narrow absorption at 1932 cm−1 (35.Li T. Quillin M.L.G.N. Phillips J. Olson J.S. Biochemistry. 1994; 33: 1433-1446Crossref PubMed Scopus (325) Google Scholar). This red shift is explained by a positive partial charge near the ligand oxygen originating from the edge of the aromatic residue. In our mutants, the replacement of Leu-29 by Tyr leads to an entirely new A3-type IR band with a dominant line at 1933 (YQR) and 1929 cm−1 (YQRF). The bulky Phe side chain at 107 appears to cause a slightly different stereochemistry of the Y and Q side chains in the distal pocket, as indicated by the additional 4 cm−1 shift to the red. The full width at half maximum of 8 and 9 cm−1 of A3 in YQR and YQRF, respectively, is comparable with that of the A0 and A1 substates in wt MbCO (17.Mourant J.R. Braunstein D.P. Chu K. Frauenfelder H. Nienhaus G.U. Ormos P. Young R.D. Biophys. J. 1993; 65: 1496-1507Abstract Full Text PDF PubMed Scopus (97) Google Scholar). In both mutants the minority species A0 displays a very broad absorption in the range between 1950 and 1980 cm−1. The high frequency implies that polar interactions between Tyr-29 and/or Gln-64 and the bound ligand are weakened or cancel each other. Possible explanations are that the Gln-64 side chain assumes an isomeric conformation in which either the nonbonded electrons of Oε are closer to the ligand or the glutamine side chain moves away from the ligand location. The width of the A0band suggests that this alternate conformation is structurally not well defined. In these experiments, the sample was illuminated for 1 s at 3 K after which the TDS experiment was started in the dark. The resulting TDS maps in the spectral regions of the bound and photodissociated CO in Fig. 3 show the absorbance change between two successive spectra, taken 200 s and 1 K apart, as a function of temperature in a contour plot. The features in the A state map (Fig. 3, A and C, top) show rebinding in the minority species, A0, taking place predominantly in the region below 40 K for both mutants. In contrast, rebinding from A3 is very different in the two mutants. In YQR, rebinding extends over the entire temperature range up to ∼110 K. The integrated absorbances of the A0 and A3 states as a function of temperature (Fig. 3B) clearly show a double-humped structure with maximum rebinding around 40 and 60 K. In the figure, we also display calculated TDS data obtained from fitting multi-gaussian barrier distributions to the TDS data, using pre-exponentials from low temperature kinetic experiments. 2A. Arcovito, M. Brunori, P. Deng, F. Draghi, A. E. Miele, O. Minkow, K. Nienhaus, and G. U. Nienhaus, manuscript in preparation. The peak enthalpies H, variances, ς, and fractional weights, f, have been compiled in Table I. In YQRF recombination from A3 is maximal at 10 K and gradually decays from there, extending up to ∼100 K. There is a substantial population rebinding below ∼50 K that can only be fitted with two barrier distributions. Moreover, the rebinding processes above 60 K, which were so apparent in YQR, appear only as shoulders in both A substates in Fig. 3D, which presents both measured and fitted integrated absorbances of the two A states as a function of temperature. Fit parameters for YQRF are also included in Table I. The observed heterogeneity in the ligand rebinding properties of A3 is also apparent in Fig. 3C, in which its TDS peak shifts to higher wave numbers with increasing temperature.Table IParameters of fits to the TDS signals integrated over the A0 and A3 substates (Fig. 3) using m
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