Structural and Dynamic Perturbations Induced by Heme Binding in Cytochrome b₅^,

Abstract

The water-soluble domain of rat hepatic cytochrome b(5) undergoes marked structural changes upon heme removal. The solution structure of apocytochrome b(5) shows that the protein is partially folded in the absence of the heme group, exhibiting a stable module and a disordered heme-binding loop. The quality of the apoprotein structure in solution was improved with the use of heteronuclear NMR data. Backbone amide hydrogen exchange was studied to characterize cooperative units in the protein. It was found that this criterion distinguished the folded module from the heme-binding loop in the apoprotein, in contrast to the holoprotein. The osmolyte trimethylamine N-oxide (TMAO) did not affect the structure of the apoprotein in the disordered region. TMAO imparted a small stabilization consistent with an unfolded state effect correlating with the extent of buried surface area in the folded region of the native apoprotein. The failure of the osmolyte to cause large conformational shifts in the disordered loop supported the view that the specificity of the local sequence for the holoprotein fold was best developed with the stabilization of the native state through heme binding. To dissect the role of the heme prosthetic group in forcing the disordered region into the holoprotein conformation, the axial histidine belonging to the flexible loop (His63) was replaced with an alanine, and the structural properties of the protein with carbon-monoxide-ligated reduced iron were studied. The His63Ala substitution resulted in a protein with lower heme affinity but nevertheless capable of complete refolding. This indicated that the coordination bond was not necessary to establish the structural features of the holoprotein. In addition, the weak binding of the heme in this protein resulted in conformational shifts at a location distant from the binding site. The data suggested an uneven distribution of cooperative elements in the structure of the cytochrome.

Insertion of the cytochrome b5 heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

“…Interestingly, multiple sets of cross-peaks were detected for the Val 61b-Gly 62b-His 63b stretch, and this segment of the protein therefore adopted at least two conformations exchanging slowly on the chemical shift time scale. The chemical shifts for one of these conformations agreed well with those previously determined for apocytochrome b 5 (Falzone et al 2001), whereas the second set of observed chemical shifts differed by approximately 0.1 ppm.…”

Structural and thermodynamic encoding in the sequence of rat microsomal cytochrome b5

“…32 In addition, the replacements F35H and H39A occurred in folded regions of the apoprotein, as indicated by dipolar contacts placing Phe35 in the proximity of His39, Val45 and Tyr74. 19 The…”

“…[9][10][11][12][13][14][15][16][17][18] The amino acid sequence of rat microsomal cyt b 5 is such that the apoprotein folds into a cooperative unit of marginal stability under physiological conditions of pH and temperature. 19 This unit comprises approximately two-thirds of the residues and corresponds closely to structural hydrophobic core 2 6 ; its DG8 of unfolding is 6 kJ mol 21 at 258C, 20 which implies that the apoprotein native state is 90% populated under those conditions. The $30 disordered residues are located in the heme binding site; once the cofactor binds, folding proceeds further, and cyt b 5 becomes a single all-encompassing cooperative unit.…”

“…The tendency of H2 to unravel first in the holoprotein contrasts with its behavior in the apoprotein, where it is a stably folded element of secondary structure. 19 The mechanisms of destabilization by chemical denaturation and by I12A replacement are clearly different. They may, however, reveal similar weaknesses in the structure.…”

Loop anchor modification causes the population of an alternative native state in an SH3‐like domain

“…As a substitute, the heme-binding loop of microsomal cytochrome b 5 (cyt b 5 ) (Fig. 2) has a suitable length (30-40 residues) and, in the apoprotein state, lacks predicted (Dunker et al 1998;Dosztanyi et al 2005) and observed (Falzone et al 1996;Bhattacharya et al 1999;Falzone et al 2001) rigid structure. In addition, a cellular environment does not force the loop to refold or collapse onto itself (Bryant et al 2005).…”