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
2LLN 2012-05-30 Solution structure of Thermus thermophilus apo-CuA
2FWL 2006-03-28 The cytochrome c552/CuA complex from Thermus thermophilus
2CUA 1999-05-28 1.6 THE CUA DOMAIN OF CYTOCHROME BA3 FROM THERMUS THERMOPHILUS
3S8G 2011-08-03 1.8 1.8 A structure of ba3 cytochrome c oxidase mutant (A120F) from Thermus thermophilus in lipid environment
3S8F 2011-08-03 1.8 1.8 A structure of ba3 cytochrome c oxidase from Thermus thermophilus in lipid environment
4G7S 2013-07-24 2.0 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236I from Thermus thermophilus
5NDC 2017-08-23 2.3 Structure of ba3-type cytochrome c oxidase from Thermus thermophilus by serial femtosecond crystallography
5U7N 2017-11-22 2.3 CRYSTAL STRUCTURE OF A CHIMERIC CUA DOMAIN (SUBUNIT II) OF CYTOCHROME BA3 FROM THERMUS THERMOPHILUS WITH THE AMICYANIN LOOP
1XME 2005-03-22 2.3 Structure of Recombinant Cytochrome ba3 Oxidase from Thermus thermophilus
2YEV 2012-05-23 2.36 Structure of caa3-type cytochrome oxidase
1EHK 2001-02-22 2.4 CRYSTAL STRUCTURE OF THE ABERRANT BA3-CYTOCHROME-C OXIDASE FROM THERMUS THERMOPHILUS
4G70 2013-07-24 2.6 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236T from Thermus thermophilus
4G7Q 2013-07-24 2.6 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236L from Thermus thermophilus
4GP5 2013-01-16 2.7 Structure of Recombinant Cytochrome ba3 Oxidase mutant Y133W from Thermus thermophilus
3EH5 2009-04-21 2.8 Structure of the reduced form of cytochrome ba3 oxidase from Thermus thermophilus
3QJV 2012-01-25 2.8 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
3QJS 2012-01-25 2.8 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
4GP4 2013-01-16 2.8 Structure of Recombinant Cytochrome ba3 Oxidase mutant Y133F from Thermus thermophilus
4GP8 2013-01-16 2.8 Structure of Recombinant Cytochrome ba3 Oxidase mutant Y133W+T231F from Thermus thermophilus
2QPE 2007-12-11 2.9 An unexpected outcome of surface-engineering an integral membrane protein: Improved crystallization of cytochrome ba3 oxidase from Thermus thermophilus
3EH4 2009-04-21 2.9 Structure of the reduced form of cytochrome ba3 oxidase from Thermus thermophilus
3QJU 2012-01-25 2.9 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
3QJQ 2012-01-25 2.9 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
4N4Y 2014-10-08 2.9 Structure of Recombinant Cytochrome ba3 Oxidase mutant G232V from Thermus thermophilus
4G71 2013-07-24 2.9 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236N from Thermus thermophilus
3QJT 2012-01-25 2.95 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
4G7R 2013-07-24 3.05 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236A from Thermus thermophilus
3EH3 2009-04-21 3.1 Structure of the reduced form of cytochrome ba3 oxidase from Thermus thermophilus
4G72 2013-07-24 3.19 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236M from Thermus thermophilus
3QJR 2012-01-25 3.2 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
2QPD 2007-12-11 3.25 An unexpected outcome of surface-engineering an integral membrane protein: Improved crystallization of cytochrome ba3 oxidase from Thermus thermophilus
3S3B 2012-05-23 3.3 Structure of Thermus thermophilus cytochrome ba3 oxidase 240s after Xe depressurization
3BVD 2008-05-20 3.37 Structure of Surface-engineered Cytochrome ba3 Oxidase from Thermus thermophilus under Xenon Pressure, 100psi 5min
3S3D 2012-05-23 3.75 Structure of Thermus thermophilus cytochrome ba3 oxidase 480s after Xe depressurization
3S3C 2012-05-23 4.0 Structure of Thermus thermophilus cytochrome ba3 oxidase 360s after Xe depressurization
3S38 2012-05-23 4.2 Structure of Thermus thermophilus cytochrome ba3 oxidase 30s after Xe depressurization
3S3A 2012-05-23 4.25 Structure of Thermus thermophilus cytochrome ba3 oxidase 120s after Xe depressurization
3S33 2012-05-23 4.45 Structure of Thermus thermophilus cytochrome ba3 oxidase 10s after Xe depressurization
3S39 2012-05-23 4.8 Structure of Thermus thermophilus cytochrome ba3 oxidase 60s after Xe depressurization

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