Document Type

Article

Publication Date

3-28-1997

Abstract

The cytochrome (cyt) b subunit of ubihydroquinone: cytochrome c oxidoreductase(bc1 complex) contains four invariant glycine (G) residues proposed to be essential for proper packing of the high and low potential (b(H) and b(L)) hemes of the bc1 complex. One of these residues, G146 located in the transmembrane helix C of cyt b of Rhodobacter capsulatus, was substituted with A and V using site-directed mutagenesis, and the effects of these substitutions on the properties of the ubiquinone oxidation (Q(o)) site and heme b(L) of the bc1 complex were analyzed. The mutants G146A and V produced properly assembled but catalytically defective bc1 complexes that are unable to support photosynthetic growth. The steady-state ubihydroquinone: cytochrome c reductase activities of the mutant complexes were about one-tenth of that of a parental strain overproducing the wild-type enzyme. Similarly, their light-activated single turnover rates were significantly lower than those of a wild-type complex. The dark potentiometric titrations revealed no significant changes in the redox midpoint potentials (E(m,7)) of the high (b(H)) and low (b(L)) potential hemes of cyt b in both G146A and V mutants. However, EPR spectroscopy of the [2Fe-2S] cluster of the bc1 complex indicated that the Q(o) site of the mutant enzymes were unoccupied. Moreover, the g(z) signal of heme b(L), but not that of heme b(H), was modified both in G146A and V, suggesting that the geometry of its ligands has been distorted. These findings indicate that this region of cyt b must be well packed around heme b(L) since even a slight increase in the size of the amino acid side chain at position 146 (such as G to A) greatly perturbs the spatial conformation of heme b(L), alters substrate accessibility and binding to the Q(o) site, and renders the bc1 complex inactive.

Publication Source (Journal or Book title)

Biochimica et Biophysica Acta - Bioenergetics

First Page

99

Last Page

108

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