Journal
FEBS JOURNAL
Volume 272, Issue 2, Pages 313-326Publisher
WILEY
DOI: 10.1111/j.1742-4658.2004.04469.x
Keywords
fumate reductase; O-site; iron-sulfur; menaquinol
Categories
Funding
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI021678] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM061606, R01GM049649] Funding Source: NIH RePORTER
- NIAID NIH HHS [AI21678] Funding Source: Medline
- NIGMS NIH HHS [GM61606, GM49649] Funding Source: Medline
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We have used fluorescence quench titrations, EPR spectroscopy and steady-state kinetics to study the effects of site-directed mutants of FrdB, FrdC and FrdD on the proximal menaquinol (MQH(2)) binding site (Q(P)) of Escherichia coli fumarate reductase (FrdABCD) in cytoplasmic membrane preparations. Fluorescence quench (FQ) titrations with the fluorophore and MQH(2) analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indicate that the Q(P) site is defined by residues from FrdB, FrdC and FrdD. In FQ titrations, wild-type FrdABCD binds HOQNO with an apparent K-d of 2.5 nm, and the following mutations significantly increase this value: FrdB-T205H (K-d = 39 nm); FrdB-V207C (K-d = 20 nm); FfdC-E29L (K-d = 25 nm); FrdC-W86R (no detectable binding); and FrdD-H80K (K-d = 20 nm). In all titrations performed, data were fitted to a monophasic binding equation, indicating that no additional high-affinity HOQNO binding sites exist in FrdABCD. In all cases where HOQNO binding is detectable by FQ titration, it can also be observed by EPR spectroscopy. Steady-state kinetic studies of fumarate-dependent quinol oxidation indicate that there is a correlation between effects on HOQNO binding and effects on the observed K-m and k(cat) values, except in the FrdC-E29L mutant, in which HOQNO binding is observed, but no enzyme turnover is detected. In this case, EPR studies indicate that the lack of activity arises because the enzyme can only remove one electron from reduced MQH(2), resulting in it being trapped in a form with a bound menasemiquinone radical anion. Overall, the data support a model for FrdABCD in which there is a single redox-active and dissociable Q-site.
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