Models for Cooperative Oxygen Binding in Hemoglobin

Equilibrium measurements of oxygen binding by 0 iron(II) and cobalt(II) picket fence porphyrins exhibit p^2, ΔΗ°, and AS° values close to those of myoglobin and cobalt myoglobin respectively. In contrast the CO affinities of simple iron(II) porphyrins are much greater than those of the hemoproteins, hemoglobin (Hb) and myoglobin (Mb). This difference is apparently caused by distal residues in Hb and Mb. With sterically constrained axial bases iron(II) and cobalt(II) picket fence porphyrins exhibit lower oxygen affinities in solution—thus modeling the "T" form of Hb. In the solid state two picket fence iron(II) porphyrins exhibit reversible cooperative oxygen binding. Recently we have isolated and fully characterized dioxygen adducts of "picket fence" iron porphyrins. Without the agency of a protein these dioxygen complexes are kinetically stable for prolonged periods as crystalline solids and for shorter periods in solution at ambient temperatures. The preparation and characterization of these analogues of the oxygen-binding hemoproteins have been recently reviewed (1). Physical properties of these model dioxygen adducts such as their Mössbauer spectra, magnetic circular dichroism spectra, v02 infrared bands, magnetic properties, and structural features about iron are closely congruent with those same features of oxyhemoglobin (Hb02)/ and oxymyoglobin (Mb02). This account summarizes oxygen binding equilibrium studies of iron and cobalt "picket fence" porphyrins and reveals the apparent relationships between the oxygen affinities of synthetic models and those of the hemoproteins. These results support a putative mechanism by which the protein globin could control oxygen affinity in the limiting "T" and "R" quaternary states of hemoglobin and thus effect cooperativity. CH-, CH3 H 3 C X I / C H 3 H 3 C \ ' / C H 3 CO y n 3 1 H 3 C v l / C H 3 Fig. 1 Picket fence porphyrin dioxygen adducts 952 JAMES P. COLLMAN and KENNETH S. SUSLICK Two types of "picket fence" porphyrins (1) have been employed in the present work. First we have used the original "picket fence" porphyrin 1_ (Fig. 1) which has a supporting axial base such as N-methyl imidazole (N-Melm) not directly attached to the porphyrin but coordinated to iron on the side opposite to the protective enclave. The second type (2) has three "pickets" on one side of the porphyrin providing a protective oxygen binding pocket and an axial imidazole bound to the opposite side of the porphyrin, 2_. Two variations of the three picket complex, having the axial imidazole joined through five and four carbon chains (2a. and 2b), are shown in Fig. 2. FePF5T FePFAT 2a FE(3-PICKET-5-C-IM) 2b FE(3-PICKET-4~C-IM) 0 II R = NHCC(CH ) Fig. 2 Three picket fence porphyrins The equilibria involving coordination of axial bases and dioxygen to synthetic porphyrins are illustrated in Fig. 3. Coordination of one axial base (eg. N-Melm) to the intermediate spin (S=l), four-coordinate iron(II) porphyrin 3̂ affords a high spin (S=2), five-coordinate complex 4̂ and is governed by the equilibrium constant Κχ. Conversion of the high spin complex 4̂ into the lowspin (S=0) six-coordinate compound 5̂ is measured by K2. Reversible coordination of the high-spin ferrous porphyrin 4_ with dioxygen affording the diamagnetic oxygen adduct 6̂ is the reaction of greatest interest. The oxygen affinity of synthetic porphyrins is measured by the magnitude of the equilibrium constant for this reaction, K3. Probably because of the spin change, for iron(II) porphyrins, K2>K^ and since these are associative equilibria this difference is magnified by lowering the temperature. Thus in the case of iron porphyrins such as _la_, the oxygen binding 5-coordinate' state 4̂ is inevitably a minor component in solution. The equilibrium described by K2 interferes with direct measurement of the oxygen affinity, K3. In principle this problem can be overcome by using an appended axial base such as that in complexes such as 2^—a technique which was employed earlier by Chang and Traylor (3). However at lower temperatures such tail-base porphyrins exhibit a strong tendency to associate by forming mixed four and six coordinate dimers (4). In the case of cobalt(II) porphyrins the high to low-spin change occurs in Κχ with the result that Ki>>K2 so that K3 can be measured directly in solution without interference from K2, even in the presence of excess axial base. With the original picket fence porphyrins the complications encountered from interfering equilibria in solution were obviated when we discovered that the Models for cooperative oxygen binding in hemoglobin 953 y < B y FE II B FE 1 1 B \ 0 F E 1 1 1 B B = N-MEIM K2>K2 B = 1,2-DIMEIM K J » K 2 Fig. 3 Equilibria involving axial bases and iron(II) porphyrins solid iron-oxygen adduct _la is porous and can be equilibrated with ambient gases. Thus under vacuum all of the iron sites in the solid complex la_ lose oxygen. When the resulting solid high-spin, five-coordinate iron complex is exposed to oxygen, equilibrium is established. Using an electronic manometer and a thermostated apparatus of known volume, the fraction of oxygenated sites, y, can be measured at different pressures of oxygen, p 0 . The solid-gas equilibrium data conform to Langmuir's isotherm (Fig. 4) which is the simple expression describing a system of independent binding sites, behaving in a non-cooperative manner. By determing p£?2 (the pressure of oxygen at half saturation; this is the reciprocal of the equilibrium constant K^) at various temperatures we were able to calculate the entropy changes associated with oxygen binding (5). The analogous solid cobalt(II) picket fence porphyrin behaves similarly, but since the competing equilibrium K2 is not significant in the case of cobalt we were also able to obtain good solution equilibrium data by determining electronic spectral changes at various partial pressures of oxygen (6) . The "tail-based" iron picket fence porphyrins 2a. and 2b_ also bind oxygen reversibly in solution at 25°C. Since in these compounds there are no competing equilibria at temperatures >0°C, it has been possible to measure oxygen affinities for â. and 21> in solution over a range of temperatures. FE + 0K3 FEOO K* = [FE021 AT P°2 [FE3 = [FE021 K3 = [FE02] [FE3+[FE0 2 ] Pn„ = M p02