Table 1.

Heme content of WT, E99 mutants and E445 mutants of E. coli cyt bd

Sample	heme b558	heme b595	excess heme b	heme d	Quinol oxidase activity (%)
WT	1	1	–	0.92	100
E445A	1	1.0	–	1.511	1
E445C	1	0.85	–	1.311	3
E445H	1	0.59	–	0.74	<3
E445Q	1	0.81	–	0.59	15
E99A	1	1.0	–	0.43	72
E99H	1	1	0.653	< 0.352	< 3
E99Q	1	1	0.633	< 0.372	< 3

¹Apparent excess heme d is attributed to perturbation of the extinction coefficients of the protein-bound heme due to the mutations. The pyridine hemochrome of heme d is not stable and quantitation by this approach is unreliable.

²The spectra of protein-bound heme d in these mutants is very perturbed making quantitation by the standard extinction coefficients unfeasible. It is assumed that the excess heme b must be bound to the site where heme d normally binds and the amount of heme d bound to that site cannot be more than that needed to account for full occupancy of that binding site.

³The amount of heme b extracted from these mutants is more than can be accounted for one heme bound to each of the two heme b binding sites (heme b₅₅₈ and heme b₅₉₅). It is assumed that each of these binding sites is fully occupied and the excess heme b binds elsewhere, presumably in the site that normally binds to heme d.