Direct measurements of ferric reductase activity of human 101F6 and its enhancement upon reconstitution into phospholipid bilayer nanodisc

Mohammed El Behery; Mika Fujimura; Tetsunari Kimura; Motonari Tsubaki

doi:10.1016/j.bbrep.2020.100730

Direct measurements of ferric reductase activity of human 101F6 and its enhancement upon reconstitution into phospholipid bilayer nanodisc

Biochem Biophys Rep. 2020 Jan 17:21:100730. doi: 10.1016/j.bbrep.2020.100730. eCollection 2020 Mar.

Authors

Mohammed El Behery¹, Mika Fujimura¹, Tetsunari Kimura¹, Motonari Tsubaki¹

Affiliation

¹ Department of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.

Abstract

We studied human 101F6 protein to clarify its physiological function as a ferric reductase and its relationship to tumor suppression activity. We found for the first time that purified 101F6 both in detergent micelle state and in phospholipid bilayer nanodisc state has an authentic ferric reductase activity by single turnover kinetic analyses. The kinetic analysis on the ferrous heme oxidation of reduced 101F6 upon the addition of a ferric substrate, ferric ammonium citrate (FAC), showed concentration-dependent accelerations of its reaction with reasonable values of K _M and V _max. We further verified the authenticity of the ferric reductase activity of 101F6 using nitroso-PSAP as a Fe²⁺-specific colorimetric chelator. 101F6 in nanodisc state showed higher efficiency for FAC than in detergent micelle state.

Keywords: Cytochrome b561; FAC; Ferric reductase activity; Human 101F6 protein; Nanodisc reconstitution; Nitroso-PSAP.