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Stabilizing roles of residual structure in the empty heme binding pockets and unfolded states of microsomal and mitochondrial apocytochrome b₅

Department of Chemistry, University of Kansas, Lawrence, Kansas 66045-7582, USA

(RECEIVED April 18, 2004; FINAL REVISION June 3, 2004; ACCEPTED June 4, 2004)

The microsomal (Mc) and mitochondrial (OM) isoforms of mammalian cytochrome b₅ are the products of different genes, which likely arose via duplication of a primordial gene and subsequent functional divergence. Despite sharing essentially identical folds, heme-polypeptide interactions are stronger in OM b₅s than in Mc b₅s due to the presence of two conserved patches of hydrophobic amino acid side chains in the OM heme binding pockets. This is of fundamental interest in terms of understanding heme protein structure–function relationships, because stronger heme–polypeptide interactions in OM b₅s in comparison to Mc b₅s may represent a key source of their more negative reduction potentials. Herein we provide evidence that interactions amongst the amino acid side chains contributing to the hydrophobic patches in rat OM (rOM) b₅ persist when heme is removed, rendering the empty heme binding pocket of rOM apo-b₅ more compact and less conformationally dynamic than that in bovine Mc (bMc) apo-b₅. This may contribute to the stronger heme binding by OM apo-b₅ by reducing the entropic penalty associated with polypeptide folding. We also show that when bMc apo-b₅ unfolds it adopts a structure that is more compact and contains greater nonrandom secondary structure content than unfolded rOM apo-b₅. We propose that a more robust -sheet in Mc apo-b₅s compensates for the absence of the hydrophobic packing interactions that stabilize the heme binding pocket in OM apo-b₅s.

Keywords: apocytochrome b₅; mitochondrion; endoplasmic reticulum; functional divergence; unfolding thermodynamics; m values; structure in the unfolded state; heme recognition

Reprint requests to: David R. Benson, Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, 2010 Malott Hall, Lawrence, KS 66045-7582, USA; e-mail: drb{at}ku.edu; fax: (785) 864-5396.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04817704.

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