Table 1. Thermodynamic parameters for Atox1-WD4 Cu⁺ transfer.

Reaction mixture	Parameter	Step 1	Step 2	Overall
Cu-Atox1C1C2 + WD4C1C2	ΔGSEC (kJ/mol)	−30.1	+24.6	−5.5
	KSEC	0.42·106 M−1	26.1·10−6 M	11.0
	ΔHITC (kJ/mol)	−25.1	+14.0	−11.1
	TΔSITC (kJ/mol)	+5.0	−10.6	−5.6
Cu-Atox1C1C2 + WD4C1A2	ΔGSEC (kJ/mol)	−26.5
	KSEC	0.09·106 M−1
	KITC	0.05·106 M−1
	ΔHITC (kJ/mol)	−52
	TΔSITC (kJ/mol)	−26
Atox1C1A2 + Cu-WD4C1C2	ΔGSEC (kJ/mol)		+30.5
	KSEC		2·10−6 M
	KITC		17·10−6 M
	ΔHITC (kJ/mol)		+52
	TΔSITC (kJ/mol)		+24

Step 1 and Step 2 relate to Fig. 1. The data for the wild type reaction, involving both Steps 1 and 2, is taken from²⁶. The mutant data comes from SEC (Fig. 2; apparent K values estimated from concentrations derived from elution profiles) and ITC (Fig. 3; K, ΔH and ΔS was determined by fitting of ITC data to a 1:1 binding model) experiments and formation of Atox1_C1C2-Cu-WD4_C1A2 corresponds to Step 1 whereas dissociation of the Atox1_C1A2-Cu-WD4_C1C2 complex corresponds to Step 2 in Fig. 1. Errors may reach 5%. See text for further details.