Linked Life Data

15654871

Source:http://linkedlifedata.com/resource/pubmed/id/15654871

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2005-1-18
pubmed:abstractText
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); FrdC-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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
1742-464X
pubmed:author
pubmed:issnType
Print
pubmed:volume
272
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
313-26
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Defining the Q-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy.
pubmed:affiliation
Department of Biochemistry, CIHR Membrane Protein Research Group, 474 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Richard.Rothery@UAlberta.ca
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.