Ligand Affinities in Hemoglobins and Their Models

The O2 affinities in biological carriers span five orders of magnitude, which at room temperature corresponds to a difference in the free energy of oxygen binding

of about 6.0 kcallmol. This wide range of O2 and CO affinities has not yet been paralleled in synthetic systems; the values for O2 affinity do not exceed those for R-state human hemoglobin. A selection of values from model systems is given in Table 4.5. 23 ,31,160-]65 For the flat-open porphyrin system (Figure 4.23) the dioxygen ostensibly binds in an unconstrained manner, but is actually subject to solvent influences. In order to obtain thermodynamic constants on these "unhindered" systems, one must gather data at several low temperatures and then extrapolate to room temperature, or obtain them from kinetic measurements, K = kon/koff, at room temperature.

For the picket-fence porphyrins, dioxygen binds in a protected pocket that is deep enough to accommodate it and to prevent the dimerization that leads to irreversible oxidation, provided that there is a slight excess of base to ensure full saturation of the coordination sites on the unprotected face of the porphyrin. 72 Thus the picket-fence, the capped, and the bis-pocket porphyrins reversibly bind dioxygen at room temperature with little oxidation over many cycles.

This stability facilitated isolation of crystals of a synthetic iron-dioxygen species of the picket-fence porphyrin. The capped porphyrin offers a more highly protected site. The low affinity these latter systems have for dioxygen indicates that the binding cavity is so small that repulsive steric interactions between coordinated dioxygen and the cap are unavoidable. The left-hand side of Figure 4.24 depicts on a logarithmic scale the range of O2 affinities. Each power of 10 corresponds to around 1.2 kcallmol at 25°C.

The right-hand side of Figure 4.24 illustrates the range of affinities for CO binding. For many synthetic systems the CO affinities are orders of magnitude greater than in the biological systems that have an O2 affinity similar to the synthetic; for example, see the entries for the picket-fence porphyrin. Comparison of the left- and right-hand sides of Figure 4.24 reveals that the strongest O2 binder, hemoglobin Ascaris, is one of the weakest CO binders. The O2 affinity of the picket-fence porphyrins is very similar to that of myoglobin, but, as will be detailed shortly, one cannot infer from this that the binding sites are strictly comparable. Indeed, similar affinities have been observed with a non-porphyrin iron complex. 121 ,162 Moreover, if the CO affinity of myoglobin paralleled that of the picket-fence porphyrins, some 20 percent of myoglobin (and hemoglobin) would be in the carbonmonoxy form (in contrast to the approximately 3 percent that occurs naturally), a level that could render reading this section while chewing gum physically taxing.

There is a convenient index to summarize the extent to which CO (or O2) binding is discriminated against for a given iron-porphyrin system. M is defined as the ratio of O2 affinity (as P1/2) to CO affinity for a particular system and experimental conditions:

From Figure 4.24 and from Tables 4.2 and 4.5 the M values calculated may be somewhat arbitrarily divided into three classes: those where M > 2 X 10 4 (good CO binder); those where 2 x 10 2 < M < 2 X 104 ; and those where M < 2 X 10 2 (good O2 binder). An analogous parameter, N, may be defined to summarize the differences in the O2 affinity between an iron-porphyrin system and its cobalt analogue:

For the picket-fence porphyrins and for vertebrate hemoglobins N is in the range 10 to 250, whereas for the flat-open porphyrins and for some hemoglobins that lack a distal histidine (e.g., hemoglobin Glycera and hemoglobin Aplysia) , N is at least an order of magnitude larger, indicating for these latter species that the cobalt analogue binds O2 relatively poorly 167,168 (see Table 4.6).

Note that whereas the O2 binding of the picket-fence porphyrins is similar to that for myoglobin, the kinetics of the process are very different; the synthetic system is more than an order of magnitude faster in kl and k_ 1 (often also referred to as kon and koff)' On the other hand, O2 binding to the pocket porphyrin is similar to that for the biological system. The factors by which ligand affinities are modulated, generally to the benefit of the organism, are subtle and varied, and their elucidation requires the precise structural information that is currently available only from x-ray diffraction experiments. Figure 4.25 shows the structural features of interest that will be elaborated upon in the next subsections.