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J. Biol. Chem., Vol. 276, Issue 22, 18968-18976, June 1, 2001
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From the Succinate-ubiquinone oxidoreductase
(SdhCDAB, complex II) from Escherichia coli is a
four-subunit membrane-bound respiratory complex that catalyzes
ubiquinone reduction by succinate. In the E. coli enzyme,
heme b556 is ligated between SdhC
His84 and SdhD His71. Contrary to a
previous report (Vibat, C. R. T., Cecchini, G., Nakamura, K.,
Kita, K., and Gennis, R. B. (1998) Biochemistry 37, 4148-4159), we demonstrate the presence of heme in both SdhC H84L and
SdhD H71Q mutants of SdhCDAB. EPR spectroscopy reveals the presence
of low spin heme in the SdhC H84L (gz = 2.92) mutant and
high spin heme in the SdhD H71Q mutant (g = 6.0). The presence of
low spin heme in the SdhC H84L mutant suggests that the heme b556 is able to pick up another ligand from the
protein. CO binds to the reduced form of the mutants, indicating that
it is able to displace one of the ligands to the low spin heme of the
SdhC H84L mutant. The g = 2.92 signal of the SdhC H84L mutant
titrates with a redox potential at pH 7.0 (Em,7) of approximately +15 mV, whereas
the g = 6.0 signal of the SdhD H71Q mutant titrates with an
Em,7 of approximately
Retention of Heme in Axial Ligand Mutants of
Succinate-Ubiquinone Oxidoreductase (Complex II) from
Escherichia coli*
§,§
Molecular Biology Division (151-S), Veterans
Affairs Medical Center, San Francisco, California 94121, the
§ Department of Biochemistry and Biophysics, University of
California, San Francisco, California 94143, and the ¶ CIHR Group
in the Molecular Biology of Membrane Proteins, Department of
Biochemistry, University of Alberta,
Edmonton, Alberta T6G 2H7, Canada
100 mV. The
quinone site inhibitor pentachlorophenol perturbs the heme optical
spectrum of the wild-type and SdhD H71Q mutant enzymes but not the SdhC H84L mutant. This finding suggests that the latter residue also plays
an important role in defining the quinone binding site of the enzyme.
The SdhC H84L mutation also results in a significant increase in the
Km and a decrease in the
kcat for ubiquinone-1, whereas the SdhD H71Q
mutant has little effect on these parameters. Overall, these data
indicate that SdhC His84 has an important role in defining
the interaction of SdhCDAB with both quinones and heme
b556.
*
This work was supported by the Department of Veterans
Affairs, National Institutes of Health Grant HL-16251, National Science Foundation Grant MCB-9728778, and a grant from the Canadian Institutes of Health Research (to J. H. W.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed:
Molecular Biology (151-S), VA Medical Center, 4150 Clement St., San
Francisco, CA 94121. Tel.: 415-752-9676; Fax: 415-750-6959; E-mail:
ceccini@itsa.ucsf.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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