Effect of redox state on the folding free energy of a thermostable electron-transfer metalloprotein: the CuA domain of cytochrome oxidase from Thermus thermophilus.


Abstract

The unfolding of the CuA domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 degrees C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of -65 kJ/mol. The corresponding value for the oxidized protein (-85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide. Study holds ProTherm entries: 9491, 9492, 9493, 9494, 9495, 9496, 9497, 9498, 9499, 9500 Extra Details: dGH2O CuA oxidized two-state model; thermodynamic cycle; reduction potentials

Submission Details

ID: U8jpUaNT3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Wittung-Stafshede P;Malmstrom BG;Sanders D;Fee JA;Winkler JR;Gray HB,Biochemistry (1998) Effect of redox state on the folding free energy of a thermostable electron-transfer metalloprotein: the CuA domain of cytochrome oxidase from Thermus thermophilus. PMID:9485471
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2YEV 2011-03-31T00:00:00+0000 2.36 Structure of caa3-type cytochrome oxidase
2CUA 1999-02-18T00:00:00+0000 1.6 THE CUA DOMAIN OF CYTOCHROME BA3 FROM THERMUS THERMOPHILUS
2FWL 2006-02-02T00:00:00+0000 0 The cytochrome c552/CuA complex from Thermus thermophilus
2LLN 2011-11-15T00:00:00+0000 0 Solution structure of Thermus thermophilus apo-CuA
1EHK 2000-02-21T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF THE ABERRANT BA3-CYTOCHROME-C OXIDASE FROM THERMUS THERMOPHILUS
1EHK 2000-02-21T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF THE ABERRANT BA3-CYTOCHROME-C OXIDASE FROM THERMUS THERMOPHILUS
1XME 2004-10-01T00:00:00+0000 2.3 Structure of Recombinant Cytochrome ba3 Oxidase from Thermus thermophilus
1XME 2004-10-01T00:00:00+0000 2.3 Structure of Recombinant Cytochrome ba3 Oxidase from Thermus thermophilus
2QPD 2007-07-23T00:00:00+0000 3.25 An unexpected outcome of surface-engineering an integral membrane protein: Improved crystallization of cytochrome ba3 oxidase from Thermus thermophilus
2QPD 2007-07-23T00:00:00+0000 3.25 An unexpected outcome of surface-engineering an integral membrane protein: Improved crystallization of cytochrome ba3 oxidase from Thermus thermophilus

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cytochrome c oxidase polypeptide I+III P98005 COX13_THET8
100.0 B Cytochrome c oxidase subunit 1 P98052 COX2_THETH
100.0 B Cytochrome c oxidase subunit 1 Q5SJ80 COX2_THET8
100.0 C Cytochrome c oxidase subunit 1 P82543 COXA_THET8
100.0 A Cytochrome c oxidase subunit 1 Q56408 COX1_THETH
100.0 A Cytochrome c oxidase subunit 1 Q5SJ79 COX1_THET8