Heme pocket

The proteins thus adapt to mutations of buried residues by changing their overall structure, which in the globins involves movements of entire a helices relative to each other. The structure of loop regions changes so that the movement of one a helix is not transmitted to the rest of the structure. Only movements that preserve the geometry of the heme pocket are accepted. Mutations that cause such structural shifts are tolerated because many different combinations of side chains can produce well-packed helix-helix interfaces of similar but not identical geometry and because the shifts are coupled so that the geometry of the active site is retained. 

Hemoglobin is a tetramer built up of two copies each of two different polypeptide chains, a- and (5-globin chains in normal adults. Each of the four chains has the globin fold with a heme pocket. Residue 6 in the p chain is on the surface of a helix A, and it is also on the surface of the tetrameric molecule (Figure 3.13). 

The A0 component is observed on reducing the pH or mutating the distal histidine residue (His64) [9a, 12]. In addition, an X-ray study of MbCO at low pH [3b] has demonstrated that His64 is far from the ligand and out of the heme pocket. On this basis the A0 state is usually associated with a protein substate in which the CO is in an apolar environment. 

The kinetics of the reaction between metMb and peroxides has attracted attention in studies that have focused on the role of the distal H64 ligand and other amino acid residues present in the distal heme pocket. Brittain et al. reported stopped-flow studies of the reaction of hydrogen peroxide with seven variants in which the distal H64 residue was replaced with a series of residues of var5fing polarity (194). Although the H64Y variant was unreactive toward peroxide, the other... 

The donor types D3, D4, and D6 of Keilin and Nicholls (37) all reduce compound I of Type A enzymes directly to the ferric state in a two-electron process without detectable intermediates. Each of these donors is probably also able to bind in the heme pocket of the free enzyme. Alcohols (type D3) form complexes with free ferric Type A enzymes whose apparent affinities parallel the effectiveness of the same alcohols as compound I donors (39). Formate (type D3) reacts with mammalian ferric enzyme at a rate identical to the rate with which it reduces compound I to free enz5mie (22). Its oxidation by compound I may thus share an initial step analogous to its complex formation with ferric enzyme. Formate also catalyzes the reduction of compound II to ferric enzyme by endogenous donors in the enz5mie (40, 41). Both compound I and compound II may thus share with the free enzyme the ability to ligate formate in the heme pocket. Nitrite, which is oxidized to nitrate by a two-electron reaction with compoimd I (type D4), also forms a characteristic complex with free enzyme (42). In both cases the reaction involves the donor in its protonated (HNO2) form. 

The donor types D2, D4, and D5 of Keilin and Nicholls (37) all reduce compound II to ferric enz5mie in a one-electron process without detectable intermediates. Donors of type D2, phenols and amines, also reduce compound I to compoimd II. Nitrite, the only member of category D4, reduces compoimd I in a two-electron step as described earlier. Donors of type D1 reduce compound I to compound II, but have no appreciable effect upon compound II itself Reactivity of the one-electron donors seems independent of heme pocket binding in the free enzyme. 

The second region is the antiparallel (3-barrel (Fig. 8) forming the core of the subunit. It includes about 250 residues from the essential histidine toward the C-terminus. The first four strands ((31-4) are contiguous and are separated from the second four strands ((35-6) by three helices ( 3-5). The first four strands form the distal side of the heme pocket and portions of the second four strands participate in binding NADPH in small subunit enzymes. 

Another significant difference between the large- and small-subunit enzymes lies in the fact that the heme d of HPII and PVC is flipped 180° relative to the heme b moiety of BLC, MLC, SCC-A, and PMC (Fig. 13). This is clearly a function of the residues that form the heme pocket, although attempts to force a change in heme orientation in HPII by mutating residues that interact with the heme were imsuccessful. The heme is situated in the (3-barrel and has interactions with the wrapping domain and with the amino-terminal arm of the R-related subunit. The dimensions of the pocket demand that heme bind in its final conformation and that flipping once inside the pocket not be possible. 

The ferro-complex CD spectrum shows that reduction of the heme iron alters the heme environment. Redox-induced protein conformation changes could alter the S5unmetry in the heme pocket or produce two binding modes for the reduced complex whose asymmetries nearly cancel each other. Redox-linked conformational changes are especially interesting in view of recent findings of oxido-reductase activity associated with the heme-hemopexin-receptor interaction (89). 

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