IscU, the scaffold protein for the
major iron–sulfur cluster
biosynthesis pathway in microorganisms and mitochondria (ISC pathway),
plays important roles in the formation of [2Fe–2S] and [4Fe–4S]
clusters and their delivery to acceptor apo-proteins. Our laboratory
has shown that IscU populates two distinct, functionally relevant
conformational states, a more structured state (S) and a more dynamic
state (D), that differ by cis/trans isomerizations about two peptidyl-prolyl peptide bonds [Kim, J.
H., Tonelli, M., and Markley, J. L. (2012) Proc. Natl. Acad.
Sci. U.S.A., 109, 454–459. Dai Z.,
Tonelli, M., and Markley, J. L. (2012) Biochemistry, 51, 9595–9602. Cai, K., Frederick, R. O.,
Kim, J. H., Reinen, N. M., Tonelli, M., and Markley, J. L. (2013) J. Biol. Chem., 288, 28755–28770].
Here, we report our findings on the pH dependence of the D ⇄
S equilibrium for Escherichia coli IscU
in which the D-state is stabilized at low and high pH values. We show
that the lower limb of the pH dependence curve results from differences
in the pKa values of two conserved histidine
residues (His10 and His105) in the two states. The net proton affinity
of His10 is about 50 times higher and that of His105 is 13 times higher
in the D-state than in the S-state. The origin of the high limb of
the D ⇄ S pH dependence remains to be determined. These results
show that changes in proton inventory need to be taken into account
in the steps in iron–sulfur cluster assembly and transfer that
involve transitions of IscU between its S- and D-states.