Glutathione-coordinated [2Fe-2S] cluster: a viable physiological substrate for mitochondrial ABCB7 transport

Abstract

The glutathione-coordinated [2Fe-2S] cluster is demonstrated to be a viable and likely substrate for physiological iron-sulfur cluster transport by Atm1p, a mitochondrial ABC export protein. Flow cytometry and colorimetric assays demonstrate a quantitative methodology for study of metal translocation proteins and their proteoliposome products.

Fig.1

Atm1p-mediated cluster transport into fluorescein-loaded proteoliposome is demonstrated by incubation in the presence of Mg-ATP. The fluorescent response is measured by flow cytometry at 530 nm, and the geometric mean of the signal indicates a decreasing kinetic profile for fluorescein within the proteoliposome as a result of the inner filter effect from [2Fe-2S] cluster, which partially absorbs the fluorescein signal within the proteoliposome. Cluster transport, along with control experiments conducted in the absence of Atm1p and Mg-ATP are denoted in black, red, and green, respectively. Controls with GSH only, GSH + Fe³⁺, and GSH + S²⁻ are shown in Figure S2.

Fig. 2

Fluorescence flow cytometry measurements of the proteoliposomes incubated with Mg-ATP (12 μM) and fluorescein-labeled glutathione [2Fe-2S] cluster (1 mM) after 1 hr at r.t. and pH 7.5, with Atm1p (left), and without Atm1p (right).

Fig.3

Concentration of iron inside the proteoliposome following 1 hr incubation with Mg-ATP and cluster. The concentration of iron was quantified by tiron coordination and comparison of absorbance values to a standard calibration curve. The final concentrations represent the results from diluted samples following work up of the liposome samples. Data, such as that shown in this plot is intended to compare relative concentrations, rather than absolute values. In fact, the absolute concentrations will be higher, but can only be estimated based on the inner proteoliposome volume. According to Geertsma et al., the total internal volume is estimated to be ~ 5 μL, and the corresponding concentration of the glutathione Fe-S cluster inside the liposome after the transport experiment is therefore estimated to be ~ 0.3 mM.