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Abstract
The overall objectives of this research project were to investigate the properties and function of late-acting iron-sulfur cluster assembly proteins in plant mitochondria and plastids. To this end, proteins from Arabidopsis thaliana (At), namely mitochondrial monothiol GRXS15, ISCA1a/1b/2 and NFU4/5 and plastidial HCF101 and NFU2, were heterologously expressed in E. coli and purified to homogeneity. The combination of UV-visible absorption and circular dichroism, electron paramagnetic resonance and resonance Raman spectroscopy was used to elucidate the nature and properties of the iron-sulfur clusters assembled on these proteins and their interactions with physiological partner proteins. The results demonstrate that GRXS15 assembles one [2Fe-2S]2+ cluster per homodimer and is an effective [2Fe-2S]2+ cluster donor for maturation of mitochondrial apo ferredoxin. Co-expression of ISCA1a/2 resulted in samples with one [2Fe-2S]2+ cluster per heterodimer, which can be converted to a form containing one [4Fe-4S]2+ cluster per heterodimer by anaerobic Fe-S cluster reconstitution. Moreover, the [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer was formed via rapid [2Fe-2S]2+ cluster transfer from [2Fe-2S]-GRXS15 to apo At ISCA1a/2, in the absence of exogenous glutathione or dithiotreitol. This result supports the proposed role of ISCA1/2 heterodimers as effectors of [2Fe-2S]2+ to [4Fe-4S]2+ cluster conversions in mitochondrial Fe-S cluster biosynthesis. Both NFU4 and NFU5 were shown to assemble one [4Fe-4S]2+ cluster per homodimer after cluster reconstitution. Moreover, ISCA1a/2, NFU4 and NFU5 were all found to be effective [4Fe-4S]2+ cluster donors for maturation of apo mitochondrial aconitase, and ISCA1a/2 was found to be a competent and efficient [4Fe-4S]2+ cluster donor for both NFU4 and NFU5. These cluster transfer processes are all rapid, unidirectional and quantitative, and demonstrate an Fe-S cluster trafficking shuttle involving GRXS15, ISCA1a/2 and NFU4 or NFU5 for the maturation of client [4Fe-4S] cluster-containing proteins in plants.
Plastidial HCF101 was shown to contain a sub-stoichiometric mixture of [4Fe-4S]2+ and linear [3Fe-4S]1+ cluster as purified under anaerobic conditions. However, as-purified HCF101 was converted to a form containing one [4Fe-4S]2+ cluster per monomer after anaerobic Fe-S cluster reconstitution. Nevertheless, the high affinity of HCF101 for binding linear [3Fe-4S]1+ clusters is evident by the observation that [4Fe-4S]2+ cluster transfer from NFU2 to apo HCF101 is a very rapid and unidirectional process, resulting in transient [4Fe-4S]-HCF101, that gradually decays with time to yield linear [3Fe-4S]1+ cluster-bound HCF101. These results, coupled with the observation that linear [3Fe-4S]1+ cluster-bound HCF101 is rapidly converted into [4Fe-4S]-HCF101 on the addition of Fe2+ ions under anaerobic conditions, raise the possibility that both cluster-bound forms of HCF101 may be present in plastids under dark and light conditions.
Plastidial HCF101 was shown to contain a sub-stoichiometric mixture of [4Fe-4S]2+ and linear [3Fe-4S]1+ cluster as purified under anaerobic conditions. However, as-purified HCF101 was converted to a form containing one [4Fe-4S]2+ cluster per monomer after anaerobic Fe-S cluster reconstitution. Nevertheless, the high affinity of HCF101 for binding linear [3Fe-4S]1+ clusters is evident by the observation that [4Fe-4S]2+ cluster transfer from NFU2 to apo HCF101 is a very rapid and unidirectional process, resulting in transient [4Fe-4S]-HCF101, that gradually decays with time to yield linear [3Fe-4S]1+ cluster-bound HCF101. These results, coupled with the observation that linear [3Fe-4S]1+ cluster-bound HCF101 is rapidly converted into [4Fe-4S]-HCF101 on the addition of Fe2+ ions under anaerobic conditions, raise the possibility that both cluster-bound forms of HCF101 may be present in plastids under dark and light conditions.