Bo Zhang , Sibali Bandyopadhyay , Priyanka Shakamuri , Sunil G. Naik , Boi Hanh Huynh ,Jérémy Couturier , Nicolas Rouhier , and Michael Kenneth Johnson

J. Am. Chem. Soc., Just Accepted Manuscript

Abstract:

Saccharomyces cerevisiae mitochondrial glutaredoxin 5 (Grx5) is the archetypical member of a ubiquitous class of monothiol glutaredoxins with a strictly conserved CGFS active-site sequence that has been shown to function in biological [Fe₂S₂]²⁺ cluster trafficking. In this work, we show that recombinant S. cerevisiae Grx5 purified aerobically after prolonged exposure of the cell-free extract to air or after anaerobic reconstitution in the presence of glutathione, predominantly contains a linear [Fe<sup>3</sub>S₄]⁺ cluster. The excited state electronic properties and ground state electronic and vibrational properties of the linear [Fe<sup>3</sub>S₄]⁺ cluster have been characterized using UV-visible absorption/CD/MCD, EPR, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic S = 5/2 linear [Fe<sup>3</sub>S₄]⁺ cluster with properties similar to those reported for synthetic linear [Fe<sup>3</sub>S₄]⁺ clusters and the linear [Fe<sup>3</sub>S₄]⁺ clusters in purple aconitase. Moreover, the results indicate that the Fe-S cluster content previously reported for many monothiol Grxs has been misinterpreted exclusively in terms of [Fe₂S₂]²⁺ clusters, rather than linear [Fe<sup>3</sub>S₄]⁺ clusters or mixtures of linear [Fe<sup>3</sub>S₄]⁺ and [Fe₂S₂]²⁺ clusters. In the absence of GSH, anaerobic reconstitution of Grx5 yields a dimeric form containing one [Fe₄S₄]²⁺ cluster that is competent for in vitro activation of apo-aconitase, via intact cluster transfer. The ligation of the linear [Fe<sup>3</sub>S₄]⁺ and [Fe₄S₄]²⁺ clusters in Grx5 has been assessed by spectroscopic, mutational and analytical studies. Potential roles for monothiol Grx5 in scavenging and recycling linear [Fe<sup>3</sub>S₄]⁺ clusters released during protein unfolding under oxidative stress conditions and in maturation of [Fe₄S₄]²⁺ cluster-containing proteins are discussed in light of these results.