Mechanism of iron release from human serum C-terminal monoferric transferrin to pyrophosphate: kinetic discrimination between alternative mechanisms

Abstract

conditions. Without additional data this small difference in the rate of decomposition does not allow us to conclude that the oxidized form further stabilizes the Cr(IV) species.O'Brien and Ozolins14 noted that at higher pH the dominant Cr(V) complexes decayed to Cr(III) products through a two-term rate law, a first-order process (first order in Cr(V); rate constant = 1.5 X 10"1 23 4s'1) and a second-order process (first order in each Cr(V) and GSH; rate constant = 9.1 X "3 M'1 s'1). At higher pH it is likely that the Cr(V) species undergoes parallel internal electron transfer and a direct reaction by a GSH molecule to form the Cr(IV) complex. This Cr(IV) complex is then rapidly reduced to Cr(III) products, in accord with our rate data showing that the second-order rate constant for the chromium(IV) decomposition (k = 0.13 M'1 s"1) in our system is at least 10 times larger than that for (k = 9.1 X 10"3 M'1 s'1) the decomposition of Cr(V) complexes.14 Acknowledgment. Funding of this research by the Kent State University Research Council is gratefully acknowledged. We thank Professors Wetterhahn and Gould for valuable discussions and Mr. Sutisak Kitareewan for helping with the recording of "time of fly" spectra during the HPLC separations. We are also grateful to reviewer no. 3 for incisive comments and helping with stylistic changes of the manuscript.

Anion-mediated Fe3+ Release Mechanism in Ovotransferrin C-lobe

“…The absence in C-oTf of SO 4 2Ϫ binding to the synergistic anion-binding groups Arg 460 -NE and -NH 2 and to Thr 461 -N can be closely related to the decelerated Fe 3ϩ release in the C-lobe. It is, however, still not clear whether the present SO 4 2Ϫ binding site corresponds to the KISAB site of the C-lobe (38). The KISAB site originally hypothesized for the C-lobe of human serum transferrin as the binding site of a variety of simple anions (38) was later investigated by the site-directed mutagenesis approach as the kinetically sensitive site toward monovalent anion, Cl Ϫ ; by this mutagenesis approach the residue Lys 569 has been assigned for the KISAB site (57).…”

“…It is, however, still not clear whether the present SO 4 2Ϫ binding site corresponds to the KISAB site of the C-lobe (38). The KISAB site originally hypothesized for the C-lobe of human serum transferrin as the binding site of a variety of simple anions (38) was later investigated by the site-directed mutagenesis approach as the kinetically sensitive site toward monovalent anion, Cl Ϫ ; by this mutagenesis approach the residue Lys 569 has been assigned for the KISAB site (57). Unlike the sulfate anion (sulfate 701) binding site in C-oTf, the residue Lys 569 exists outside the interdomain cleft.…”

“…The differential rate for the anion-mediated Fe 3ϩ release for the two lobes are observed in a wide range of pH, including neutral pH. The mode of anion effects on the Fe 3ϩ release kinetics is also different for the two lobes; a KISAB proposed for the C-lobe is absent in the N-lobe (38). The original model proposed by Bates and co-workers (49,53) (54 -56), no attempt has been made to directly compare the isolated N-and C-lobes with respect to the anion-dependent Fe 3ϩ release kinetics and the crystal structure of the anionbinding site.…”

“…When the functional characteristics are compared for the Nand C-lobes of transferrins, the effects of anions on the Fe 3ϩ release kinetics are strikingly different for the two lobes. The rate for the anion-mediated Fe 3ϩ release is much slower from the C-lobe than from the N-lobe (35-37), and a kinetically significant anion-binding (KISAB) 1 site that has been proposed for the C-lobe as the binding site of a variety of simple anions is not kinetically detected for the N-lobe (38). Detailed structural and functional comparisons between the N-and C-lobes of transferrins may provide crucial information about the hinge motion mechanism of biologically important multidomain proteins.…”

Structural and functional consequences of removal of the interdomain disulfide bridge from the isolated C‐lobe of ovotransferrin

“…Kinetically, there was no difference between the two protein forms for the pyrophosphate-induced iron release: both the forms exhibited monophasic time courses and similar k obs values as a function of anion concentrations. Previously, the accelerating effect of chloride was observed with the iron release from hTf-Fe C~K retchmar & Raymond, 1986;Egan et al, 1992;Zak et al, 1997!. Likewise, the addition of chloride accelerated the pyrophosphate-induced iron release process of C-oTf and~2CM 8SS!-C-oTf; the extent of the acceleration was almost the same for the two protein forms. The iron release mechanism of C-oTf seems to be distinctly different from that of N-oTf in which iron release kinetics follows a In an earlier study of the full-length oTf, the affinity of iron to the Cys478-Cys671 reduced C-lobe was reported to be lower than to the disulfide-intact counterpart~Williams et al, 1985!.…”