Table III. Heme characteristics and oxygenase activities of PADOX-1 with mutations in putative axial heme ligands.

Soluble recombinant GST-PADOX-1s with mutations in either the putative proximal heme ligand (His-389) or the putative distal heme ligand (His-163) were purified by affinity chromatography before their absorbance spectra were recorded, and their oxygenase activity was measured with linoleate. Activities were normalized to the amount of recombinant protein using immunoblot densities. K_d values for cyanide binding were calculated from absorbance increases near 430 nm as a function of the cyanide concentration, as done for the experiment in Fig. 7. K_i values were calculated from the decreases in activity as a function of cyanide concentration, as for the experiment in Fig. 9.

Construct	Resting Soret peak position	Oxygenase specific activity	Kd for CN−	Ki for CN−
	nm	% wild type	mm	µm
Wild type	413	100	13 ± 2	5 ± 1
H163C	414	6	1.6 ± 0.3	40 ± 5
H163M	420	17	>280a	127 ± 11
H163Q	414	<1	>240a	NDb
H163Y	414	12	66 ± 12a	18 ± 2
Y386F	413	<1	11 ± 1	NDb
R387H	414	99	15 ± 2	31 ± 6
H389C	400	8	75 ± 10a	32 ± 4
H389M	396	8	60 ± 6a	30 ± 4
H389Q	Nonec	Nonec
H389Y	Nonec	Nonec

^aNo isosbestic point observed (not a simple E + CNS⁻ ⇆ E-CNS⁻ transition).

^bNot determined.

^cRecombinant protein expressed in insoluble form.