Fluorescence lifetime values of purified hetero-dimer^a.

Protein	τ 1 (ns)	τ 2 (ns)	τ 3 (ns)	A 1 (%)	A 2 (%)	A 3 (%)	χ 2	τ av b (ns)
GFPK25C	2.92 ± 0.06	—	—	100	—	—	1.05 ± 0.00
apoCyt b562N80C–GFPK25C	2.93 ± 0.01			99.9 ± 0.05			1.00 ± 0.03
Reconstituted dimerc	0.28 ± 0.08	2.92	—	93.7 ± 0.26	6.3 ± 0.16	—	0.99 ± 0.01
Cyt b562A100C–GFPK25C	0.16 ± 0.04	1.35 ± 0.01	2.92	91.7 ± 0.05	4.90 ± 0.12	3.78 ± 0.52	1.09 ± 0.01	0.32
Cyt b562H63C–GFPK25C	0.20 ± 0.01	2.92	—	93.7 ± 5.42	6.31 ± 5.42	6.31 ± 5.42	0.97 ± 0.00	0.37
Cyt b562N80C–GFPK25C	0.31 ± 0.03	1.53 ± 0.26	2.92	97.5 ± 0.79	2.50 ± 0.44	—	1.07 ± 0.05	0.49
Cyt b562K15C–GFPK25C	0.56 ± 0.03	2.17 ± 0.33	2.92	92.9 ± 1.76	5.44 ± 0.88	1.68 ± 0.09	1.10 ± 0.07	0.70
GFPS174C	2.88 ± 0.02			100			1.02 ± 0.00
Cyt b562A100C–GFPS174C	0.15 ± 0.00	2.88	—	99.8 ± 0.01	0.22 ± 0.02	—	1.06 ± 0.15	0.15
Cyt b562H63C–GFPS174C	0.12 ± 0.02	1.67 ± 0.10	2.88	89.8 ± 6.56	6.60 ± 4.25	3.59 ± 2.37	0.98 ± 0.12	0.32
Cyt b562N80C–GFPS174C	0.10 ± 0.00	0.75 ± 0.39	2.88	99.2 ± 0.07	0.77 ± 0.07	—	1.06 ± 0.02	0.11
Cyt b562K15C–GFPS174C	0.23 ± 0.01	2.82 ± 0.05	2.88	80.5 ± 1.57	16.7 ± 1.50	2.76 ± 0.15	0.97 ± 0.02	0.74

^aConditions: [protein] = 20 μM, 1 mL, prepared in 0.1 M potassium phosphate buffer pH 7.0 at 25 °C. Fluorescence lifetimes were evaluated by bi- or tri-exponential decay model. Values expressed are means ± SD of three parallel measurements.

^bThe intensity average lifetimes were calculated using eqn (1).

^cHetero-dimer formed by apoCyt b₅₆₂^N80C–GFP^K25C and then reconstituted by heme.