Metal ion coordination in 'R' and 'T' state hybrid hemoglobins as revealed by optical, EPR and sulphhydryl reactivity studies
TL;DR: In this paper, the sulphhydryl environment in various mixed-metal hybrid hemoglobins, viz. α2(Cu)-β2(FeCO), α2-FeCO)-β 2(Cu), α 2-Cu-β2-Ni, α 2(Ni)- β2-Cu, β 2-Ni-β 2 (β2)-β 4-PDS, was studied by reacting them with the 4,4′-dithiodipyridine (4PDS) reagent.
Abstract: The sulphhydryl environment in various mixed-metal hybrid hemoglobins, viz. α2(Cu)-β2(FeCO), α2(FeCO)-β2(Cu), α2(Cu)-β2(Ni), α2(Ni)-β2(Cu), was studied by reacting them with the sulphhydryl reagent, 4,4′-dithiodipyridine (4-PDS). The reactivity was compared with that of HbCO, NiHb and CuHb. It is found that there exists a correlation between conformational change and metal ion environment, not only at the extreme R and T states but also the intermediate conformations. EPR examinations of these hybrids show that both in R state-[Cu(II)-Fe(II)] and T state-[Cu(II)-Ni(II)] hybrids at neutral pH and in the absence of IHP, CuPPIX, irrespective of the subunit in which it is present, has a mixed-metal ion environment: Species 1, a five-coordinated Cu2+ complex with strong proximal histidine bond and species 2, a four-coordinated complex without any covalent linkage with Ne F8-histidine.
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TL;DR: The bond between N(epsilon) and Fe is fundamental to the structure-function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO.
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TL;DR: Study of the effect of inositol hexaphosphate on the three low spin ferrous compounds of hemoglobin with O2, CO, and NO shows that the R leads to T transition causes either a rupture or at least a very dramatic stretching of the bond from the iron to the heme-linked histidine, such that an equilibrium is set up between five- and six-coordinated hemes.
Abstract: Studies of high spin ferrous and ferric derivatives led us to conclude that in the quaternary R structure the state of the hemes is similar to that in the free alpha and beta subunits, but in the T structure a tension acts on the hemes which tries to pull the iron and the proximal histidine further from the plane of the porphyrin. We have now studied the effect of inositol hexaphosphate (IHP) on the three low spin ferrous compounds of hemoglobin with O2, CO, and NO. IHP failed to switch the quaternary structure of carbonmonoxy- and oxyhemoglobin A to the T state, but merely caused a transition to an as yet undefined modification of the R structure. IHP is known to cause a switch to the T structure in hemoglobin Kansas. We have found that this switch induces red shifts of the visible alpha and beta absorption bands and the appearance of a shoulder on the red side of the alpha band; these changes are very weak in carbonmonoxy- and slightly stronger in oxyhemoglobin Kansas. As already noted by previous authors, addition of IHP to nitrosylhemoglobin A induces all the changes in uv absorption and CD spectra, sulfhydryl reactivities, and exchangeable proton resonances normally associated with the R leads to T transition, and is accompanied by large changes in the Soret and visible absorption bands. Experiments with nitrosyl hybrids show that these changes in absorption are caused predominantly by the hemes in the alpha subunits. In the accompanying paper Maxwell and Caughey (J. C. Maxwell and W. S. Caughey (1976), Biochemistry, following paper in this issue) report that the NO in nitrosylhemoglobin without IHP gives a single ir stretching frequency characteristic for six-coordinated nitrosyl hemes; addition of IHP causes the appearance of a second ir band, of intensity equal to that of the first, which is characteristic for five-coordinated nitrosyl hemes. Taken together, these results show that the R leads to T transition causes either a rupture or at least a very dramatic stretching of the bond from the iron to the heme-linked histidine, such that an equilibrium is set up between five- and six-coordinated hemes, biased toward five-coordinated hemes in the alpha and six-coordinated ones in the beta subunits. The reason why IHP can switch nitrosyl-, but not carbonmonoxy- or oxyhemoglobin A, from the R to the T structure is to be found in the weakening of the iron-histidine bond by the unpaired NO electron and by the very short Fe-NO bond length.
219 citations
TL;DR: It is concluded that the two quaternary forms of hemoglobin can be observed for the halfligated states of hemochemistry and that the switch between the two forms is responsible for the co-operativity.
118 citations
TL;DR: The oxygen affinity of the alpha and beta subunits may be regulated by different mechanisms.
Abstract: Resonance Raman spectra have been obtained of the alpha deoxy and beta deoxy subunits within valency hybrid hemoglobins both in the high-affinity (R) and low-affinity (T) structures Upon conversion from the R to the T structure, the vibrational frequency of the Fe(II)-N epsilon(His-F8) bond changes from 223 to 207 or 203 cm-1 in the alpha deoxy subunit and from 224 to 220 or 217 cm-1 in the beta deoxy subunit We estimate that the Fe(II)-N epsilon(His-F8) bond is stretched by the R leads to T transition 3 times more in the alpha subunit (0024 A) than in the beta subunit (00085 A) and, accordingly, the strain energy developed in that bond is 8 times larger in the alpha than in the beta subunit Hence, the oxygen affinity of the alpha and beta subunits may be regulated by different mechanisms
115 citations
TL;DR: There is a large difference in the carbon dioxide binding constants of the β chain α-amino group in the oxy and deoxy forms of human hemoglobin, and that 2,3-diphosphoglycerate suppresses this difference, probably by binding strongly to the βChain α-AMino group of deoxyhemoglobin and displacing any bound carbon dioxide.
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86 citations