TABLE 1.

Hemoglobin and haptoglobin affinities of IsdH^N1

	MetHb binding		Hp binding
Protein	Kda	-Fold decreaseb	Kda	-Fold decrease
	nm		nm
Wild-type IsdHN1c	17 ± 10		35 ± 10
B1-B2 loop
N120A	21 ± 2.4	1.2	46 ± 4.1	1.3
T123A	80 ± 2.4	4	45 ± 3.7	1.3
Q124A	62 ± 3.3	3	24 ± 5.3	0.7
Y125A	2287 ± 1.2	134	1025 ± 0.4	21
Y126A	2607 ± 6.9	153	No binding
H127A	1230 ± 0.5	72	No binding
F128A	720 ± 13	42	No binding
F129A	698 ± 2.6	41	No binding
B3-B4 loop
N151A	24 ± 1.0	1.4	33 ± 16	0.9
S153A	219 ± 6.4	11	46 ± 2.5	1.3
T154A	203 ± 16	10	29 ± 1.7	0.8
B7-B8 loop
D205A	53 ± 3.7	3	53 ± 10	1.5
D206A	55 ± 4.1	3	57 ± 12	1.6
G207A	32 ± 7.5	2	20 ± 7	0.6
E208A	70 ± 7.6	4	24 ± 12	0.7
E209A	55 ± 8.0	3	33 ± 15	0.9
Surrounding mutants
V112A	20 ± 2.4	1.2	50 ± 1.0	1.4
D133A	24 ± 7.5	1.4	52 ± 1.5	1.5
D136A	24 ± 1.0	1.4	37 ± 5.6	1.0
K141A	20 ± 1.2	1.2	55 ± 14	1.6
K142A	16 ± 6.9	1.0	47 ± 16	1.3

^aDissociation constants were measured twice using SPR on a Biacore system. The reported errors are the average fits from the average dissociation constant.

^bThe -fold decrease is the affinity of the mutant that is compared to the wild-type protein affinity for its substrate.

^cThe wild-type protein consists of residues 86-229 of the IsdH protein connected at its N terminus to a 19-residue histidine tag. Each mutant is identical to the wild-type protein but contains a single alanine residue substitution at the indicated position.