Document Detail


Close proximity of Cys64 and Cys140 in the delta subunit of Escherichia coli F1-ATPase.
MedLine Citation:
PMID:  8307987     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The delta subunit of the F1-ATPase from Escherichia coli contains 2 cysteine residues, one at position 64 and the second at position 140 of the amino acid sequence. These residues were specifically labeled with sulfhydryl reagents in this study without labeling other -SH groups in the enzyme. Modification of Cys140 by maleimides such as N-ethylmaleimide or fluorescein maleimide resulted in a reconstitutively active enzyme that was indistinguishable from the native protein. Labeling of Cys64 with or without concomitant labeling of Cys140 resulted in a reconstitutively inactive enzyme. The ATPase activity of either form of the labeled enzyme was unaffected. However, labeling of Cys64 was accompanied by dissociation of the delta subunit from the enzyme. These observations suggest a role for the microenvironment of Cys64 in interactions of the delta subunit with other subunits in the enzyme. Two types of evidence support the conclusion that the 2 cysteine residues of the delta subunit are in close proximity. First, incorporation of pyrene maleimide into both delta cysteines led to excimer formation. Second, incubation of F1 with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in quantitative formation of a disulfide bond between Cys64 and Cys140, presumably via disulfide interchange. The enzyme containing the internally cross-linked delta subunit exhibited an undiminished ability to support proton pumping when reconstituted into F1-depleted membrane vesicles. The presence of 2 closely apposed cysteinyl residues in the delta subunit of the native enzyme places constraints on the type of structure that may be proposed for the subunit.
Authors:
M Ziegler; R Xiao; H S Penefsky
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  269     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1994 Feb 
Date Detail:
Created Date:  1994-03-17     Completed Date:  1994-03-17     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  4233-9     Citation Subset:  IM    
Affiliation:
Department of Biochemistry and Molecular Biology, State University of New York, Health Science Center at Syracuse 13210.
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MeSH Terms
Descriptor/Qualifier:
Cysteine / chemistry
Escherichia coli / enzymology
Membrane Proteins / metabolism
Proton-Translocating ATPases / chemistry,  metabolism,  ultrastructure*
Spectrometry, Fluorescence
Structure-Activity Relationship
Sulfhydryl Reagents / chemistry
Grant Support
ID/Acronym/Agency:
GM21737/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Membrane Proteins; 0/Sulfhydryl Reagents; 52-90-4/Cysteine; EC 3.6.3.14/Proton-Translocating ATPases

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