A kinetic model for R- and T-state hemoglobin. Flash photolysis of heme-imidazole-carbon monoxide mixtures

For almost a decade heme model compounds have been designed to test the influence of proximal base restraint or of distal steric hindrance upon the ligand affinity of hemoglobins. Despite the variety of molecular structures which have been successively proposed, the evaluation of the reported data is rendered difficult because of the small number of examples available within each series. In this paper we report on the kinetics of binding of oxygen and carbon monoxide with a series of nine closely related heme models. The 'basket-handle porphyrins' allow one to modify the constraints exerted upon a chelated proximal base as well as the chemical environment of the distal side of the heme. One salient feature of these models is the possibility of introducing a hydrogenbond stabilization of the oxygen by incorporating an amide group in the vicinity of the iron centre. The structural changes among models are sufficiently 'soft' to cause an almost continuous variation of the binding constants and rate parameters. The latter are found to obey a definite linear free energy relationship which proves that the series is homogenous from a thermodynamic viewpoint. This suggests an alternative way for comparing the trends in ligand binding in different heme model families with those of heme proteins, which is developed in the discussion using literature dataThe original concepts put forward by Perutz [I, 21 have greatly stimulated the investigations of heme model compounds capable of simulating certain aspects of hemoglobin reactivity. The central problem deals with the existence of a high-affinity form (R) and a low-affinity form (T) of hemoglobin and with the mechanism by which they are converted into each other. The absence of a protein structure in model compounds obviously precludes the elucidation of the trigger mechanism as such, but the idea that steric constraint of the proximal base may lead to reduced ligdnd affinities has been substantiated 13 -81.A more controversial question is concerned with the possibility that distal side steric hindrance might also serve to regulate ligand binding to the heme. The extent of steric hindrance is believed to be larger with linear (CO) than with bent (02) ligands and this process has been proposed to account principally for the kinetic difference between ligands in hemoglobin [9 -1 I].Scveral groups have designed model porphyrins to test either ( o r both) hypotheses. In most cases an extraneous 'proximal' base had to be supplied to the porphyrin in solution, although the advantages of a covalently attached internal proximal base had been recognized earlier [12, 131. In a recent comprehensive review of the various heme niodels, Jameson and Ibers [I41 have discussed CO and O 2 affinities in the light of stereochemical data. One of their Ahh/c~i.itr/iori.s. Unless otherwise stated, the shorthand expression BHP is to be understood as iron(I1) complexes of 'basket-handle porphyrins' 1-9 [18-201. Details concerning the structure of the othcr model compounds discussed in the text ca...

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“…The validity ofEqns (8,9) in describing O2 and CO binding with the ether-BHPs and amide-BHPs is apparent in Fig. 3.…”

Section: Discussionmentioning

confidence: 79%

“…Fortunately, in most situations of interest, the following linear relationship has been found to hold [9]: where z may be considered as representing the fraction of the transition state. Eqns (4-7) can be combined to give the integral relations: (8) and (9 ) where a and k , are specific for both reaction partners.…”

Section: Discussionmentioning

confidence: 99%

Linear free-energy relationships in binding of oxygen and carbon monoxide with heme model compounds and heme proteins

et al. 1984

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“…For these studies a suspension of -3% hematocrit of erythrocytes or reconstituted ghosts containing hemoglobin and albumin as well as spectrin, was prepared in 300 mosmol PBS. Immediately after resuspension to minimize settling, these cells were added to an Aminco-Morrow stop-flow analysis apparatus connected to a microprocessor, a large computer memory, and a cathode ray tube display (36). The cell suspension was then very rapidly diluted by an equal volume of50 mosM saline to produce a final osmolarity of 175 mosmol.…”

Section: Methodsmentioning

confidence: 99%

Reconstitution of spectrin-deficient, spherocytic mouse erythrocyte membranes.

Shohet¹

1979

J. Clin. Invest.

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A B S T R A C T To study directly the role of spectrin in erythrocyte membrane function, we have designed a reconstituted membrane system using erythrocyte membranes from spectrin-deficient mice and purified spectrin from normal mice. The normal spectrin is inserted into the spectrin-deficient spherocytes by exchange hemolysis. Thereafter, raising the ionic strength and temperature reseals the cells and, with time, facilitates binding of the spectrin to the spectrindeficient membranes. The binding is apparently specific as shown by its dependence upon the concentration of undenatured spectrin and the concentration of salt used, as well as by the immunofluorescent appearance of the reconstituted cells after treatment with specific antispectrin antibody.In terms of in vitro cellular behavior, the reconstituted preparations show marked changes in comparison to the untreated spherocytes. In particular, membrane stability, as measured by the reduction of myelin figure formation and lipid loss, is considerably enhanced. In addition, membrane fusion, which occurs readily with the untreated spherocytes, is virtually eliminated. Finally, the osmotic behavior of the native spherocytes is appreciably altered, such that the early phase of osmotically induced swelling, as measured in a highspeed stop-flow apparatus, is delayed and modified.Taken together, these findings indicate specific roles for spectrin in the stabilization of the erythrocyte membrane, in the limitation of membrane fusion, and in the modulation of the membrane's response to osmotic stress.

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“…The dynamics associated with ligand binding to the heme group has been studied for many years in both native and mutant proteins [3][4][5][6][7][8][9][10][11][12][13][14][15] and in heme model compounds. [16][17][18][19][20][21][22][23][24] The binding process for model compounds involves at least two steps: the diffusion of ligand through the surrounding solvent or buffer followed by the bond formation step between the diatomic ligand and the iron. In proteins, the process is complicated by the protein matrix where various entry and exit channels, as well as docking sites and cavities within the protein, lead to a complexity in the overall reaction process that is still not fully understood.…”

Section: Introductionmentioning

confidence: 99%

“…18,23 However, for such compounds, the CO ligand escape rate is much larger than its geminate rebinding rate, so the latter process is difficult or impossible to detect. 23 Moreover, in the absence of an added imidazole ligand, the CTAB encapsulated heme samples again display complex kinetic evolution, probably due to a base elimination mechanism 18,42 (i.e., upon CO photolysis, there is a delayed dissociation of the weak proximal base before CO rebinds). The problems mentioned above have made clear kinetic comparisons between the imidazole ligated and unligated species problematic.…”

Section: Introductionmentioning

confidence: 99%

CO Rebinding to Protoheme: Investigations of the Proximal and Distal Contributions to the Geminate Rebinding Barrier

et al. 2005

J. Am. Chem. Soc.

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The rebinding kinetics of CO to protoheme (FePPIX) in the presence and absence of a proximal imidazole ligand reveals the magnitude of the rebinding barrier associated with proximal histidine ligation. The ligation states of the heme under different solvent conditions are also investigated using both equilibrium and transient spectroscopy. In the absence of imidazole, a weak ligand (probably water) is bound on the proximal side of the FePPIX-CO adduct. When the heme is encapsulated in micelles of cetyltrimethylammonium bromide (CTAB), photolysis of FePPIX-CO induces a complicated set of proximal ligation changes. In contrast, the use of glycerol-water solutions leads to a simple two-state geminate kinetic response with rapid (10-100 ps) CO recombination and a geminate amplitude that can be controlled by adjusting the solvent viscosity. By comparing the rate of CO rebinding to protoheme in glycerol solution with and without a bound proximal imidazole ligand, we find the enthalpic contribution to the proximal rebinding barrier, H p , to be 11 ± 2 kJ/mol. Further comparison of the CO rebinding rate of the imidazole bound protoheme with the analogous rate in myoglobin (Mb) leads to a determination of the difference in their distal free energy barriers: ΔG D ≈ 12 ± 1 kJ/mol. Estimates of the entropic contributions, due to the ligand accessible volumes in the distal pocket and the xenon-4 cavity of myoglobin (~3 kJ/mol), then lead to a distal pocket enthalpic barrier of H D ≈ 9 ± 2 kJ/mol. These results agree well with the predictions of a simple model and with previous independent room-temperature measurements (Tian et al. Phys. Rev. Lett. 1992, 68, 408) of the enthalpic MbCO rebinding barrier (18 ± 2 kJ/mol).

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A kinetic model for R- and T-state hemoglobin. Flash photolysis of heme-imidazole-carbon monoxide mixtures

Cited by 119 publications

References 5 publications

Linear free-energy relationships in binding of oxygen and carbon monoxide with heme model compounds and heme proteins

Linear free-energy relationships in binding of oxygen and carbon monoxide with heme model compounds and heme proteins

Reconstitution of spectrin-deficient, spherocytic mouse erythrocyte membranes.

CO Rebinding to Protoheme: Investigations of the Proximal and Distal Contributions to the Geminate Rebinding Barrier

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