Principles of Protein Structure - G. Schultz 1982

Protein-ligand interactions
Heme binding sites
Chemical transformations of the heme iron atom within the protein microenvironment

In Hemoglobin, the Fe²⁺ ion is protected from oxidation by a nonpolar environment. The polypeptide chain of the heme-containing protein provides a specialized microenvironment for its prosthetic groups, facilitating specific chemical transformations while suppressing unwanted Side Reactions. The functional importance of this microenvironment is clearly illustrated by Hemoglobins. In an aqueous medium, ferroheme binds O₂, but is simultaneously oxidized to ferriheme, which is incapable of reversible molecular oxygen binding [637]. However, heme can reversibly bind oxygen without oxidation when embedded in a nonpolar medium, such as benzene, where the extraction of an electron from the ferrous ion is significantly more difficult than in Water [639, 647]. It can thus be concluded that The primary function of the nonpolar heme-binding pocket is to protect the ferrous state of heme from oxidation by shielding it from water.

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Fig. 10.2. Chemical analogue of the hemoglobin active center [727].

Protoporphyrin IX is located at the center of the scheme. One propionic side chain (top right) forms a peptide bond with the analogue of the peripheral His of hemoglobin, which is linked to polyethylene glycol (PEG) via a Gly residue. The other propionic group (bottom right) is attached via a spacer chain to imidazole, representing the analogue of the proximal His. This chemical O₂ carrier is qualitatively similar to hemoglobin in its pattern of reversible molecular oxygen binding and in certain spectral properties.

Chemical model of compounds possessing hemoglobin-like activity. The Synthesis of the hemoglobin active-site model relies on substituting the protein with another polymer [639, 727] (Fig. 10.2) or with hydrophobic side chains attached to the heme to form a "picket fence" [797]. Model systems of this kind can be valuable for elucidating Structure–function relationships in natural macromolecules. Furthermore, they allow the principles of protein structural Organization to be extrapolated to conditions that are incompatible with the functional integrity of the native Proteins (unusual temperatures, pH values, and solvent compositions).

Fig. 10.3. Structural changes in hemoglobin upon oxidation. a — Possible positions of the iron atom relative to the porphyrin plane (shaded) in deoxyhemoglobin [804, 654] and oxyhemoglobin [550, 651]. Center-to-center distances are taken from studies cited by Phillips [690]. It should be noted that the heme geometry in oxyhemoglobin was determined using so-called picket-fence complexes [651], i.e., model compounds. The positions of two porphyrin nitrogen atoms (N_p) and the N_ε atom of the proximal His are indicated. Upon oxidation, the N_ε atom shifts by I_d—I₀ = 0.6±0.1 Å. b — Conformational Changes in the hemoglobin subunit upon oxidation (Perutz, private communication). The Displacement of the F helix toward the H helix leads to the extrusion of the penultimate residue of the β-subunit, Tyr HC2, from the pocket between the two helices. This radically alters THE POSITION OF the carboxyl group of the terminal His HC3 residue; Val FG5 corresponds to Val-98.

O₂ and CO bind to hemoglobin as bent ligands. This binding geometry appears to play a key role in The Mechanism of CO discrimination. The affinity of hemoglobins for O₂ is twice as low as that of simple iron-porphyrin models, such as heme dissolved in benzene. In contrast, the CO-binding capacities of these two systems differ by a factor of 100 [648]. Consequently, the protein-formed microenvironment discriminates against CO without significantly altering its ability to bind O₂. This selectivity is based, in part, on the distinct electronic effects exerted on CO and O₂ by the trans-position of the second axial iron Ligand [649], the so-called proximal His (Fig. 10.2).

However, another aspect appears to be more significant. Both in the simple iron-porphyrin system [650, 651] and in hemoglobin [690], molecular oxygen binds as a bent ligand (Fig. 10.3, a), in which form it is best described as an O₂^- superoxide ([652, 653]; compare, however, [650]). Conversely, CO binds linearly in the simple system [648, 654], whereas it binds in a bent conformation in all hemoglobins studied to date [655–657]. Thus, the protein matrix housing the heme dictates a binding geometry that is favorable for O₂ and unfavorable for CO. It is worth noting that despite this discrimination, the affinity of hemoglobins for CO remains 500-fold higher than for O₂ [634].

The evolution of ligand binding toward a geometry unfavorable for CO was presumably driven by the need to counteract endogenous CO released during the Catabolism of Porphyrins into Bile pigments [85]. Were it not for this protective mechanism, the metabolically generated CO could occupy approximately one-third of all hemoglobin binding sites [649]. The current view on CO discrimination is that hemoglobins, which emerged more than 10⁸ years ago, have undergone adaptation to conditions that might otherwise have proved lethal — namely, tobacco smoking and air pollution.

In Cytochromes, the heme iron atom is protected from undesired ligands. Within cytochromes, the protein microenvironment serves two Functions: it shields the heme from adventitious ligands and maintains the Redox Potential of the iron atom at the appropriate level. The protective role of Met-80, which coordinates the heme iron atom in cytochrome c (Fig. 7.8), has been previously suggested [509]. In the absence of this interaction, the iron atom is readily and uncontrollably reduced, for instance, by ascorbate [658]. It can be argued that the polypeptide chain ensures The fidelity of electron transfer along its biological pathway [509]. The unusual ligand inertness of the iron ion in cytochrome b₅ [297] is likewise attributed to the rigidity of its axial ligands (Section 10.3).

The redox potential of the heme iron atom in cytochrome c is determined by its axial ligands. The Influence of the apoprotein on the redox potential was investigated by Moore and Williams [659]. Their study compared the redox potentials of heme-model compounds [660] and utilized structural data from various cytochromes along with nuclear magnetic Resonance data for distantly related c-type cytochromes. The authors concluded that the redox potential of the heme iron is dictated by its axial ligands. For example, two Histidine side chains acting as axial ligands, as found in b₅, confer a lower redox potential than the histidine–Methionine pair typically present in c-type cytochromes. When comparing c-type cytochromes, variations in the Fe–S bond length are observed [659]. A shortening of this bond by 0.1 Å is associated with a 400 mV decrease in the redox potential, reflecting an increase in the electron-donating capacity of the methionine sulfur atom.