1
Anaerobic MicroScale Thermophoresis
Application Note NT-MO-026
Anaerobic MicroScale Thermophoresis reveals the Redox
dependency of ferredoxin in mitochondrial Fe/S biogenesis
Sven-Andreas Freibert
1,2
, Holger Webert
1
, Nina Schlinck
3
, Roland Lill
1,2,4
1
Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Straße 6, 35032 Marburg, Germany
2
LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans-Meerwein-Str., 35043 Marburg, Germany
3
NanoTemper Technologies GmbH, Flößergasse 4, 81369 München, Germany
4
Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str. 10, 35043 Marburg, Germany
Abstract
Maturation of iron-sulphur (Fe/S) proteins
involves a complex biosynthetic machinery.
Here we investigate the interaction of one
component, Yah1, with its interaction partners
apo-Isu1 and Nfs1-Isd11. Using anaerobic
MicroScale Thermophoresis (MST) we show
the redox dependency of the interaction
between Yah1 and Isu1 as well as between
Yah1 and Nfs1-Isd11. Reduced but not
oxidized Yah1 tightly interacts with apo-Isu1
indicating a dynamic interaction between
Yah1−apo-Isu1.
Introduction
Iron-sulphur clusters (Fe/S) are one of the oldest
inorganic prosthetic groups on earth. They play
important roles in fundamental cellular processes
such as respiration, amino acid and cofactor
metabolism, DNA metabolism and repair, and the
regulation of environmental responses
2-4
. The
mitochondrial ISC (iron-sulphur cluster assembly)
machinery is crucial for the synthesis of all Fe/S
clusters (mitochondrial, cytosolic and nuclear) as
well as for cellular iron regulation. Mutations in
many mitochondrial ISC genes cause
neurodegenerative, haematological and metabolic
diseases with complex clinical and biochemical
phenotypes
5,6-8
.
The first step of Fe/S protein maturation in yeast
crucially requires the scaffold protein Isu1, the
cysteine desulfurase complex Nfs1-Isd11
9
and
the [2Fe-2S] ferredoxin Yah1 (Fig. 1)
10-14
. So far,
the exact nature of the interactions within the
machinery has not been resolved in detail.
Figure 1: NMR solution structure of the yeast ferredoxin
Yah1 (PDB: 2MJE), in its reduced state and with its flexible C-
terminus. The [2Fe-2S] cluster is represented as stick model.
Isu1-interacting residues are in sphere-representation.