Thermal stability effects of removing the type-2 copper ligand His306 at the interface of nitrite reductase subunits.
Nitrite reductase (NiR) is a highly stable trimeric protein, which denatures via an intermediate, N(3)<--(k)-->U(3)--(k)-->F (N-native, U-unfolded and F-final). To understand the role of interfacial residues on protein stability, a type-2 copper site ligand, His306, has been mutated to an alanine. The characterization of the native state of the mutated protein highlights that this mutation prevents copper ions from binding to the type-2 site and eliminates catalytic activity. No significant alteration of the geometry of the type-1 site is observed. Study of the thermal denaturation of this His306Ala NiR variant by differential scanning calorimetry shows an endothermic irreversible profile, with maximum heat absorption at T (max) approximately equal to 85 degrees C, i.e., 15 degrees C lower than the corresponding value found for wild-type protein. The reduction of the protein thermal stability induced by the His306Ala replacement was also shown by optical spectroscopy. The denaturation pathway of the variant is compatible with the kinetic model N(3)--(k)-->F(3), where the protein irreversibly passes from the native to the final state. No evidence of subunits' dissociation has been found within the unfolding process. The results show that the type-2 copper sites, situated at the interface of two monomers, significantly contribute to both the stability and the denaturation mechanism of NiR.
Stirpe
Andrea
A
Dipartimento di Fisica e Unità CNISM, Università della Calabria, Ponte P. Bucci, Cubo 31C, 87036 Arcavacata di Rende, Cosenza, Italy.
Sportelli
Luigi
L
Wijma
Hein
H
Verbeet
Martin Ph
MP
Guzzi
Rita
R
D000409:Alanine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
D001426:Bacterial Proteins; D008024:Ligands; D021122:Protein Subunits; D011994:Recombinant Proteins; D006639:Histidine; D003300:Copper; D009572:Nitrite Reductases; D000409:Alanine
Biophys Chem. 1997 Dec 1;69(2-3):125-35
17029925
Biochemistry. 2002 Jan 22;41(3):1060-9
11790130
Int J Biol Macromol. 1999 Nov;26(2-3):129-34
10517519
Biochemistry. 2001 Apr 17;40(15):4622-32
11294629
J Biol Chem. 1995 Nov 17;270(46):27458-74
7499203
Science. 1991 Jul 26;253(5018):438-42
1862344
Org Biomol Chem. 2004 Jul 21;2(14):1953-6
15254616
Biochemistry. 2003 Apr 15;42(14):4075-83
12680761
Annu Rev Biophys Biophys Chem. 1990;19:159-88
2194474
Anal Biochem. 1995 Mar 20;226(1):80-4
7785783
FEBS Lett. 2005 Sep 26;579(23):5223-6
16165132
J Gen Microbiol. 1993 Apr;139(4):725-33
8515232
Biophys Chem. 1998 Mar 9;70(3):247-57
9546202
Inorg Chem. 2004 Nov 29;43(24):7591-3
15554622
Biochemistry. 2002 Jun 18;41(24):7573-85
12056888
J Biol Chem. 2000 Aug 4;275(31):23957-64
10811642
Biopolymers. 2006 Dec 5;83(5):487-97
16881076
Biochemistry. 1988 Mar 8;27(5):1648-52
3365417
Biochemistry. 1995 Mar 14;34(10):3286-99
7880824
Biochem Biophys Res Commun. 1992 Sep 30;187(3):1529-35
1329738
Biophys J. 1992 Apr;61(4):921-35
19431826
Biochemistry. 1995 Sep 26;34(38):12107-17
7547950
Bioorg Med Chem. 2005 Sep 1;13(17):5013-20
15993087
Biochim Biophys Acta. 2001 Jun 11;1547(2):397-407
11410296
Biochemistry. 2004 Oct 5;43(39):12563-74
15449946
Biochim Biophys Acta. 2005 Aug 31;1752(1):47-55
16085470
Annu Rev Biophys Biomol Struct. 2000;29:105-53
10940245
Methods Enzymol. 1987;154:511-33
3323816
Protein Eng. 2002 Jul;15(7):575-84
12200540
J Biol Chem. 1999 Sep 24;274(39):27711-6
10488113
J Biol Chem. 1997 Nov 7;272(45):28455-60
9353305
J Mol Biol. 2005 Jul 8;350(2):300-9
15927201
Biochemistry. 1986 Sep 23;25(19):5432-44
3778869