Quaternary transformation induced changes at the heme in deoxyhemoglobins (original) (raw)
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Proceedings of the National Academy of Sciences, 1979
Absence of heme-localized strain in T state hemoglobin: Insensitivity of heme-imidazole resonance Raman frequencies to quaternary structure (protein dynamics/cooperativity/strain energy/heme vibrations) ABSTRACT Substitution of pentadeuterated 2-methylimidazole in (2-methylimidazole)Fe(II)protoporphyrin IX, a model complex for deoxyHb, shifts three bands in the low-frequency resonance Raman spectrum 380 --373 cm-l, 348 -345 cm-1, and 220 -_ 218 cm-'. The first of these is assigned primarily to Fe-imidazole stretching, and the other two are assigned to porphyrin deformation modes with substantial Fepyrrole stretching contributions. The three bands are observed in deoxyHb and Mb. The Fepyrrole modes are at essentially the same frequencies in the two proteins, but the Fe-imidazole mode is 6 cm-l lower in deoxyHb than Mb, implying a slight alteration in the heme-imidazole linkage. No change greater than 2 cm-l is observed when Hb Kempsey is switched from the R to the T state. This observation places an upper limit on the energy stored in the Fe-imidazole bond of T state deoxyHb, which is estimated to be <0.2 kcal/mol (<836.8 J/mol). The iron-imidazole bond is a key chemical link in heme proteins, in most of which at least one of the heme axial ligands is an imidazole side chain. For hemoglobin (Hb) and myoglobin (Mb), the iron-imidazole bond is the only point of covalent attachment of the heme prosthetic group. Along with the noncovalent contacts, it supports the structural changes (1) that are known to accompany the binding of 02 to the five-coordinate iron atom in deoxyHb (2) and Mb (3). Moreover, the forces that induce the quaternary structural change that accompanies 02 binding to deoxyHb must be transmitted at least in part through the iron-imidazole linkage (4). Accordingly, it would be valuable to have available a probe of the iron-imidazole bond strength. The vibrational frequency associated with stretching of this bond should provide a suitable probe, because bond strengths and force constants are directly correlated. Resonance Raman spectroscopy is capable of monitoring vibrational modes associated with the heme group (5). The most intense resonance Raman bands arise from porphyrin ring vibrations (6). Resonance enhancement is much weaker for the vibrational modes associated with the iron atom.
Biopolymers, 2006
In an attempt to gain further insight into the nature of the low frequency vibrational modes of hemoglobin and its isolated subunits, a comprehensive study of several different isotopically labeled analogues has been undertaken and is reported herein. Specifically, the resonance Raman spectra, between 200 and 500 cm -1 , are reported for the deoxy and ligated (CO and O 2 ) forms of the isolated a and b subunits containing the natural abundance or various deuterated analogues of protoheme. The deuterated protoheme analogues studied include the 1,3,5,8-C 2 H 3 -protoheme (d12-protoheme), the 1,3-C 2 H 3 -protoheme (1,3-d6-protoheme), the 5,8-C 2 H 3 -protoheme (5,8-d6-protoheme), and the meso-C 2 H 4 -protoheme (d4-protoheme). The entire set of acquired spectra has been analyzed using a deconvolution procedure to help correlate the shifted modes with their counterparts in the spectra of the native forms. Interestingly, modes previously associated with so-called vinyl bending modes or propionate deformation modes are shown to be quite sensitive to deuteration of the peripheral methyl groups of the macrocycle, shifting by up to 12-15 cm -1 , revealing their complex nature. Of special interest is the fact that shifts observed for the 1,3-d6-and 5,8-d6-protoheme analogues confirm the fact that certain modes are associated with a given portion of the macrocycle; i.e., only certain modes shift upon deuteration of the 1 and 3 methyl groups, while others shift upon deuteration of the 5 and 8 methyl groups. Compared with the spectra previously reported for the corresponding myoglobin derivatives, the data reported here reveal the appearance
Biochemistry, 2008
Resonance Raman spectroscopy is employed to characterize heme site structural changes arising from conformational heterogeneity in deoxyMb and ligated derivatives; i.e., the ferrous CO (MbCO) and ferric cyanide (MbCN) complexes. The spectra for the reversed forms of these derivatives have been extracted from the spectra of reconstituted samples. Dramatic changes in the low frequency spectra are observed, where newly observed RR modes of the reversed forms are assigned using protohemes that are selectively deuterated at the four methyl groups or at the four methine carbons. Interestingly, while substantial changes in the disposition of the peripheral vinyl and propionate groups can be inferred from the dramatic spectral shifts, the bonds to the internal histidyl imidazole ligand and those of the Fe-CO and Fe-CN fragments are not significantly affected by the heme rotation, as judged by lack of significant shifts in the ν(Fe-N His), ν(Fe-C) and ν(C-O) modes. In fact, the apparent lack of an effect on these key vibrational parameters of the Fe-N His , Fe-CO and Fe-CN fragments is entirely consistent with previously reported equilibrium and kinetic studies that document virtually identical functional properties for the native and reversed forms. Conformational heterogeneity involving rotational disorder of the heme about the α-γ meso axis (Figure 1) in native and reconstituted myoglobins was first studied by NMR spectroscopy and circular dichroism (CD) spectroscopy in the 1980's. 1-8 This conformational heterogeneity has been found to occur not only in the reconstituted metMb form, 3 but also in the native deoxy 2 and ligated forms; i.e., MbCO. 3 At equilibrium, the reversed conformation still persists in very small amounts for aquo-metMb (~ 4% for HH Mb 24 and ~8% for SW Mb 3) and deoxy Mb, MbCO and metMb-CN (~8% for SW Mb) 2,3 while for the recently discovered met neuroglobin (metNgb) an ~70/30 ratio has been reported. 8 Most of these studies focused only on the detection of the reversed orientation and the effects of temperature, pH, spin states or heme peripheral substituents on the extent and kinetics of heme reorientation, the NMR studies detecting and assigning only the shifted resonances of the heme methyl protons. 3 In later NMR relaxation studies, however, the structural consequences of heme orientation were investigated by analyzing the distance between the heme iron and protons of the Ile FG5 C γ H and Phe CD1 C ζ H side chains of sperm whale Mb (SW Mb). 9,10 These studies showed that the heme is slightly displaced away from Ile FG5 C γ H in the reversed form. Though apparently displaced within the heme pocket, recent studies employing the so-called normal coordinate structural decomposition methods suggest that the protein matrices of both the native and reversed forms induce similar types of distortions on heme macrocyclic core. 11
Inorganic chemistry, 2016
We analyzed the oxygen (O2) and carbon monoxide (CO) binding properties, autoxidation reaction rate, and FeO2 and FeCO vibrational frequencies of the H64Q mutant of sperm whale myoglobin (Mb) reconstituted with chemically modified heme cofactors possessing a variety of heme Fe electron densities (ρFe), and the results were compared with those for the previously studied native [Shibata, T. et al. J. Am. Chem. Soc. 2010 , 132 , 6091 - 6098 ], and H64L [Nishimura, R. et al. Inorg. Chem. 2014 , 53 , 1091 - 1099 ], and L29F [Nishimura, R. et al. Inorg. Chem. 2014 , 53 , 9156 - 9165 ] mutants in order to elucidate the effect of changes in the heme electronic structure and distal polar interaction contributing to stabilization of the Fe-bound ligand on the functional and vibrational properties of the protein. The study revealed that, as in the cases of the previously studied native protein [Shibata, T. et al. Inorg. Chem. 2012 , 51 , 11955 - 11960 ], the O2 affinity and autoxidation reacti...
Spectroscopic markers of the T<--> R quaternary transition in human hemoglobin
Biophysical chemistry, 2005
In this work, we use a sol-gel protocol to trap and compare the R and T quaternary states of both the deoxygenated (deoxyHb) and carbonmonoxide (HbCO) derivatives of human hemoglobin. The near infrared optical absorption band III and the infrared CO stretching band are used to detect the effect of quaternary structure on the spectral properties of deoxyHb and HbCO; comparison with myoglobin allows for an assessment of tertiary and quaternary contributions to the measured band shifts. The RX T transition is shown to cause a blue shift of the band III by~35 cm À1 for deoxyHb and a red shift of the CO stretching band by only~0.3 cm À1 for HbCO. This clearly shows that quaternary structure changes are transmitted to the heme pocket and that effects on deoxyHb are much larger than on HbCO, at least as far as the band energies are concerned. Experiments performed in the ample temperature interval of 300-10K show that the above quaternary structure effects are bstaticQ and do not influence the dynamic properties of the heme pocket, at least as probed by the temperature dependence of band III and of the CO stretching band. The availability of quaternary structure sensitive spectroscopic markers and the quantitative measurement of the quaternary structure contribution to band shifts will be of considerable help in the analysis of flash-photolysis experiments on hemoglobin. Moreover, it will enable one to characterize the dynamic properties of functionally relevant hemoglobin intermediates and to study the kinetics of both the TYR and RYT quaternary transitions through time-resolved spectroscopy. D
We have examined the Fe2±-NE(HisF8) complex in hemoglobin A (HbA) by measuring the band profile of its Raman-active VFeHiS stretching mode at pH 6.4, 7.0, and 8.0 using the 441-nm line of a HeCd laser. A line shape analysis revealed that the band can be decomposed into five different sublines at £1 = 195 cm-1, 12 = 203 cm-1, £3 = 212 cm-1, Q4= 218 cm-1, and f5 = 226 cm-1 . To identify these to the contributions from the different subunits we have reanalyzed the VFe-His band of the HbA hybrids a(Fe)2f3(Co)2 and a(Co)2f3(Fe)2 reported earlier 133-216). Moreover we have reanalyzed other Raman bands from the literature, namely the VFe-HiS band of the isolated hemoglobin subunits a SH_ and 3SH-HbA, various hemoglobin mutants (i.e., Hb(Tyrc7at-Phe), Hb(TyrC7a--His), Hb M-Boston and Hb M-lwate), N-ethylmaleimide-des(Arg141 c) hemoglobin (NES-des(Arg141 t)HbA) and photolyzed carbonmonoxide hemoglobin (Hb*CO) measured 25 ps and 10 ns after photolysis. These molecules are known to exist in different quaternary states. All bands can be decomposed into a set of sublines exhibiting frequencies which are nearly identical to those found for deoxyhemoglobin A.
Conformational changes in hemoglobin triggered by changing the iron charge
In this work the hemoglobin conformational changes induced by changing the iron charge have been studied and compared with Myoglobin. Mössbauer spectroscopy was used to follow the change of the iron conformation. In order to compare the conformational relaxation of hemoglobin and myoglobin, and to study a possible influence of the quaternary structure, an intermediate metastable state of hemoglobin has been created by low temperature X-ray irradiation of methemoglobin. The irradiation reduces the Fe(III) of the heme groups to Fe(II) Low Spin, where the water is still bound on the sixth coordination. Heating cycles performed at temperatures from 140 K to 200 K allow the molecules to overcome an activation energy barrier and to relax into a stable conformation such as deoxy-hemoglobin or carboxy-hemoglobin, if CO is present. Slightly different structures (conformational substates) reveal themselves as a distribution of energy barriers (ΔG#). The distribution of the activation energy, for the decay of the Fe(II) Low Spin intermediate, has been fitted with a Gaussian. For comparison, published myoglobin data were re-analysed in the same way. The average energy value at characteristic temperature is very similar in case of myoglobin and hemoglobin. The larger Gaussian energy distribution for myoglobin with respect to hemoglobin shows that more conformational substates are available. This may be caused by a larger area exposed to water. In hemoglobin, part of the surface of the chains is not water accessible due to the quaternary structure.
Journal of the American Chemical Society, 2012
Encapsulation of hemoglobin (Hb) in silica gel preserves structure and function, but greatly slows protein motion, thereby providing access to intermediates along the allosteric pathway that are inaccessible in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet laser excitation provides probes of heme reactivity and of key tertiary and quaternary contacts. These probes were monitored in gels after deoxygenation of oxyHb and after CO binding to deoxyHb, which intiate conformational change in the R-T and T-R directions, respectively. The spectra establish that quaternary structure change in the gel takes a week or more, but that the evolution of heme reactivity, as monitored by the Fe-histidine stretching vibration, νFeHis, is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving νFeHis frequencies span the full range of values between those previously associated with the high-and low-affinity end states, R and T. This result supports the tertiary two-state (TTS) model, in which the Hb subunits can adopt high-and low-affinity tertiary structures, r and t, within each quaternary state. The spectra also reveal different tertiary pathways, involving the breaking and re-formation of E and F inter-helical contacts in the R-T direction but not the T-R direction. In the latter, tertiary motions are restricted by the T quaternary contacts. * Corresponding author: spiro@chem.washington.edu .