Heme-hemopexin reduction

E. Reduction of Heme-Hemopexin and Binding of Exogenous Ligands... 

Exposure of ferri-heme-hemopexin to imidazole or KCN can displace one or both of the heme coordinating His residues, but millimolar concentrations are required (138). Other potential ligands such as azide or fluoride are inactive. This coordination stability of the ferri-heme-hemopexin bis-histidyl complex, despite the exposed heme site, is home out by thermal imfolding studies (Section IV,F). Reduction of the heme-hemopexin complex, however, has dramatic effects on its stability. 

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). 

Fig. 12. Schematic views of bis-histidyl ferri-, ferro-, and CO-ferro-heme-hemopexin. Unlike myoglobin with one open distal site, heme bound to hemopexin is coordinated to two strong field ligands, either of which a priori may be displaced by CO. This may well produce coupled changes in protein conformation like the Perutz mechanism for 02-binding by hemoglobin (143). The environment of heme bound to hemopexin and to the N-domain may be influenced by changes in the interactions of porphyrin-ring orbitals with those of aromatic residues in the heme binding site upon reduction and subsequent CO binding.

It is not known whether iron-transferrin affects hemopexin receptor expression. However, the presence of iron transferrin attenuates the induction of MT-1 mRNA by heme-hemopexin in hepatoma cells, and ferric citrate is even more effective (L. Sung, M. Shibata, P. J. Morales, N. Shipulina, A. Smith, unpublished results). This suggests that the process of iron uptake into cells, which has been linked to electron transport for reduction of the iron, utilizes cofactors required for the hemopexin system. 

If both MT-1 and HO-1 mRNA induction by heme-hemopexin involves a copper-redox enzyme in both heme transport (and consequent induction of HO-1 mRNA) and the signaling pathway for MT-1 expression, a plausible working model can be formulated by analogy with aspects of the yeast iron uptake processes and with redox reactions in transport (Figure 5-6). First, the ferric heme-iron bound to hemopexin can act as an electron acceptor, and reduction is proposed to be required for heme release. The ferrous heme and oxygen are substrates for an oxidase, possibly NADH-dependent, in the system for heme transport. Like ferrous iron, ferrous heme is more water soluble than ferric heme and thus more suitable as a transport intermediate between the heme-binding site on hemopexin and the next protein in the overall uptake process. The hemopexin system would also include a copper-redox protein in which the copper electrons would be available to produce Cu(I), either as the copper oxidase or for Cu(I) transport across the plasma membrane to cytosolic copper carrier proteins for incorporation into copper-requiring proteins [145]. The copper requirement for iron transport in yeast is detectable only under low levels of extracellular copper as occur in the serum-free experimental conditions often used. 

Tm 48°C respectively). This is consistent with a reduction step aiding heme release by weakening the complex prior to, or during, the actual release of heme effected by the hemopexin receptor. (The nondestructive mode of transport by hemopexin with recycling of intact protein demands mechanisms to reversibly lower the affinity of the complex.) With or without NaCl, lowering the pH from 7.4 to 6.5 has little effect... 

Fig. 14. Effects of temperature on the absorbance of hemopexin and the N-domain of hemopexin. The unfolding of hemopexin and N-domain in 25 mM sodium phosphate, pH 7.4, was examined using absorbance spectroscopy (N. Shipulina et al., unpublished). The second derivative UV absorbance spectra of the protein moieties were used to follow protein unfolding and the Soret and visible region spectra to monitor the integrity of the heme complexes, as done with cytochrome 6502 (166). The ferri-heme complex is more stable than the apo-protein moiety, but the is slightly lower than that assessed by DSC, indicating that changes in conformation occur before thermodynamic unfolding. Reduction causes a large decrease in heme-complex stabihty, which is proposed to be a major factor in heme release from hemopexin by its cell membrane receptor, and addition of 150 mM sodium chloride enhanced the stabihty of ah forms of hemopexin.

The chemistry and biochemistry of Hpx has been reviewed and a crystal structure is available. Hemopexin is present in serum at about 10 pM and its primary function is to transport released heme to its degradation site in the parenchymal cells of the liver via receptor-mediated endocytosis. Encapsulation of a single heme by Hpx occurs via bis-histidyl protein side-chain coordination of the Fe. Spectroelectrochemical investigation of the heme-Hpx assembly gives insight into the role of Hpx in controlling the reduction potential of the heme Fe, the efficiency of electron transfer at the metal centre, the influence of bis-histidyl coordination at the Fe centre, and the possible role of Fe redox in the Hpx-mediated transport and recycling of heme. 

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