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PEDS Advance Access originally published online on October 24, 2005
Protein Engineering Design and Selection 2005 18(12):571-579; doi:10.1093/protein/gzi067
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform

Na Sun, An Wang, Aaron B. Cowley, Adriana Altuve, Mario Rivera and David R. Benson1

Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045-7582, USA

1 To whom correspondence should be addressed. E-mail: drb{at}ku.edu

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the McS71L b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins.

Keywords: apoprotein/cytochrome b5/heme replacement/mutagenesis/stability

Received May 27, 2005; revised July 26, 2005; accepted September 1, 2005.

Edited by Michael Hecht


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L. Wang, A. B. Cowley, and D. R. Benson
Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform
Protein Eng. Des. Sel., October 24, 2007; (2007) gzm053v1.
[Abstract] [Full Text] [PDF]


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