Iron is an essential element for all photosynthetic organisms. The biological use of this transition metal is as an enzyme cofactor, predominantly in electron transfer and catalysis. The main forms of iron cofactor are, in order of decreasing abundance, iron-sulfur clusters, heme, and di-iron or mononuclear iron, with a wide functional range. In plants and algae, iron-sulfur cluster assembly pathways of bacterial origin are localized in the mitochondria and plastids, where there is a high demand for these cofactors. A third iron-sulfur cluster assembly pathway is present in the cytosol that depends on the mitochondria but not on plastid assembly proteins. The biosynthesis of heme takes place mainly in the plastids. The importance of iron-sulfur cofactors beyond photosynthesis and respiration has become evident with recent discoveries of novel iron-sulfur proteins involved in epigenetics and DNA metabolism. In addition, increased understanding of intracellular iron trafficking is opening up research into how iron is distributed between iron cofactor assembly pathways and how this distribution is regulated.

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we release the intact complex largely devoid of detergent This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.Membrane proteins perform a wide range of biological functions including respiration, signal transduction and molecular transport. Despite their obvious importance, these proteins and their complexes remain notoriously difficult to study. High-resolution methods, such as X-ray crystallography, require recombinant expression and crystallization of proteins, both of which are difficult for membrane subunits. Low-resolution methods to characterize membrane protein complexes include blue native gel electrophoresis 1 , sedimentation equilibrium or sedimentation velocity measurements via analytical ultracentrifugation 2 and small-angle X-ray scattering 3 . Although excellent results regarding the size of membrane proteins can be obtained, large quantities of protein are usually required, and it is essential to account for the contribution of the micelle to the overall mass. It can also be difficult to obtain definitive data on large or unstable complexes that do not form single oligomeric states in detergent solutions. Moreover the low accuracy of ultracentrifugation or X-ray scattering data does not normally reveal lipid binding or allow observation of posttranslational modifications.

Background: ABC transporters of mitochondria (ATM) are required for formation of cytosolic iron-sulfur clusters and molybdenum cofactor.Results: Arabidopsis ATM3 and yeast Atm1 transport radiolabeled glutathione disulfide (GSSG). Transport of glutathione trisulfide (GS-S-SG) was demonstrated by mass spectrometry.Conclusion: A mitochondrial transporter exports glutathione polysulfide.Significance: Identification of substrate(s) of ATMs defines their role in metal cofactor assembly and iron homeostasis.