Table 1.

The steady-state kinetic parameters, coenzyme and substrate binding constants and rates of hydride and proton transfer for wild-type POR and variant enzymes. All rates and binding constants were measured at 25 °C. The rates of hydride and proton transfer were measured from the average of at least five time dependent absorption measurements by laser excitation of similar levels of ternary enzyme-substrate complex and by following absorbance changes at 696 nm. The amplitude of the absorbance change at 696 nm is also shown.

Enzyme	kcat (s− 1)	KdNADPH (nM)	KdPchlide (μM)	khydride (× 106 s− 1)	kproton (× 104 s− 1)	ΔmAbs 696 nm
Wild-type	0.17 ± 0.002	21 ± 1	5.6 ± 0.6	2.21 ± 0.06	2.72 ± 0.04	102 ± 1
S16C	0.16 ± 0.001	325 ± 90	3.5 ± 0.5	2.11 ± 0.03	2.68 ± 0.03	61 ± 3
G19A	0.16 ± 0.001	182 ± 31	3.5 ± 0.4	2.08 ± 0.07	2.67 ± 0.02	92 ± 2
R38V	0.16 ± 0.001	411 ± 76	5.4 ± 0.6	2.12 ± 0.03	2.69 ± 0.01	54 ± 4
N39V	0.02 ± 0.001	23 ± 6	28.9 ± 1.4	1.98 ± 0.02	2.71 ± 0.03	10 ± 0.4
K42A	0.16 ± 0.001	230 ± 81	4.5 ± 0.4	2.14 ± 0.09	2.67 ± 0.04	85 ± 3
N90A	0.07 ± 0.002	94 ± 7	21.4 ± 0.6	2.28 ± 0.04	3.17 ± 0.04	31 ± 2
Y94F	0.17 ± 0.003	63 ± 4	11.8 ± 0.7	2.23 ± 0.02	3.11 ± 0.01	80 ± 1
T145A	0.01 ± 0.001	33 ± 2	39.7 ± 2.1	n. d.	n. d.	2 ± 0.2
T147S	0.02 ± 0.003	60 ± 3	6.5 ± 0.7	n. d.	n. d.	2 ± 0.2
T147F	0.01 ± 0.003	117 ± 3	10.3 ± 0.6	n. d.	n. d.	1 ± 0.1
N149V	0.01 ± 0.003	55 ± 2	19.2 ± 1.2	n. d.	n. d.	2 ± 0.2
S189A	0.16 ± 0.001	38 ± 16	4.9 ± 0.4	2.15 ± 0.07	2.69 ± 0.01	69 ± 4
T230A	0.16 ± 0.001	181 ± 16	11.9 ± 1.4	1.63 ± 0.07	2.26 ± 0.03	51 ± 3
T230S	0.16 ± 0.001	48 ± 17	4.6 ± 0.5	1.71 ± 0.06	2.38 ± 0.06	60 ± 1
T230F	0.01 ± 0.001	436 ± 87	44.9 ± 3.5	n. d.	n. d.	2 ± 0.2
H236A	0.10 ± 0.004	30 ± 11	9.4 ± 0.6	2.18 ± 0.03	2.72 ± 0.01	33 ± 1