Reconstitution, characterization, and [2Fe–2S] cluster exchange reactivity of a holo human BOLA3 homodimer

Wachnowsky, Christine; Rao, Brian; Sen, Sambuddha; Fries, Brian; Howard, Cecil J.; Ottesen, Jennifer J.; Cowan, J. A.

doi:10.1007/s00775-019-01713-x

Cited by 3 publications

(3 citation statements)

References 56 publications

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“…Interestingly, holo‐BOLA1 (reconstituted by itself, without addition of apo‐GLRX5) transferred its cluster to ferredoxins (Fig. 7), similar to our observation with holo‐BOLA3 [20]. However, BOLA1‐GRX5 was found unable to donate or accept cluster from either ISCA1 or ISCA2 (data not shown), as summarized in Table 1.…”

Section: Resultssupporting

confidence: 85%

“…The reaction mixture was argon-purged for 30 min, and then, Fe 3+ and L-cysteine were added to a final concentration of 0.6 mM each. The reaction mixture was passed through a PD-10 desalting column after incubating at room temperature for 1.5 h. The Bradford assay was used to estimate the total concentration of apo-proteins, and holo-concentrations were determined by extinction coefficients and confirmed by iron quantitation [20]. Reconstitution of holo-GLRX5 by itself was performed in the presence of glutathione (without BOLA1) by a similar method [25,27].…”

Section: In Vitro Reconstitution Of Apo-proteinsmentioning

confidence: 99%

“…Reconstitution of holo-GLRX5 by itself was performed in the presence of glutathione (without BOLA1) by a similar method [25,27]. Holo-BOLA1 (without GLRX5) was also prepared by similar method [20]. Reconstitution of ISCU, ISCA1, and ISCA2 was performed by incubating 200 lM of apo-protein with 50 mM DTT and 3 M urea, with argon purging for 30 min, and then making up to 1 mM in Fe 3+ and 1 mM in S 2À .…”

Section: In Vitro Reconstitution Of Apo-proteinsmentioning

confidence: 99%

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Cluster exchange reactivity of [2Fe‐2S]‐bridged heterodimeric BOLA1‐GLRX5

2020

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Mitochondrial BOLA1 is known to form a [2Fe‐2S] cluster‐bridged heterodimeric complex with mitochondrial monothiol glutaredoxin GLRX5; however, the function of this heterodimeric complex is unclear. Some reports suggest redundant roles for BOLA1 and a related protein, BOLA3, with both involved in the maturation of [4Fe‐4S] clusters in a subset of mitochondrial proteins. However, a later report on the structure of BOLA1‐GLRX5 heterodimeric complex demonstrated a buried cluster environment and predicted a redox role instead of the cluster trafficking role suggested for the BOLA3‐GLRX5 heterodimeric complex. Herein, we describe a detailed kinetic study of relative cluster exchange reactivity involving heterodimeric complex of BOLA1 with GLRX5. By the use of CD spectroscopy, it is demonstrated that [2Fe‐2S]‐bridged BOLA1‐GLRX5 can be readily formed by cluster uptake from donors such as ISCU or [2Fe‐2S](GS)4 complex, but not from ISCA1 or ISCA2. Rapid holo‐formation following delivery from [2Fe‐2S](GS)4 supports possible physiological relevance in the cellular labile iron pool. Holo [2Fe‐2S] BOLA1‐GLRX5 heterodimeric complex is incapable of donating cluster to apo protein acceptors, providing experimental support for a nontrafficking role. Finally, we report the formation and reactivity of the holo [2Fe‐2S]‐bridged BOLA1 homodimer (lacking a partner GLRX). While the holo‐heterodimer is thermodynamically more stable, by contrast the holo BOLA1 homodimer does demonstrate facile cluster exchange reactivity.

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Section: Resultssupporting

confidence: 85%

Section: In Vitro Reconstitution Of Apo-proteinsmentioning

confidence: 99%

Section: In Vitro Reconstitution Of Apo-proteinsmentioning

confidence: 99%

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Cluster exchange reactivity of [2Fe‐2S]‐bridged heterodimeric BOLA1‐GLRX5

2020

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Spectroscopic and functional characterization of the [2Fe–2S] scaffold protein Nfu from Synechocystis PCC6803

et al. 2022

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Biochemical impact of a disease-causing Ile67Asn substitution on BOLA3 protein

et al. 2021

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Iron-sulfur (Fe-S) cluster biosynthesis involves the action of a variety of functionally distinct proteins, most of which are evolutionarily conserved. Mutations in these Fe-S scaffold and trafficking proteins can cause diseases such as multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalopathy. Herein, we investigate the effect of Ile67Asn substitution in the BOLA3 protein that results in the MMDS2 phenotype. Although the exact functional role of BOLA3 in Fe-S cluster biosynthesis is not known, the [2Fe-2S]-bridged complex of BOLA3 with GLRX5, another Fe-S protein, has been proposed as a viable intermediary cluster carrier to downstream targets. Our investigations reveal that the Ile67Asn substitution impairs the ability of BOLA3 to bind its physiological partner GLRX5, resulting in a failure to form the [2Fe-2S]-bridged complex. Although no drastic structural change in BOLA3 arises from the substitution, as evidenced by wild type and mutant BOLA3 1H-15N HSQC and IM-MS experiments, this substitution appears to influence cluster reconstitution on downstream proteins leading to the disease phenotype. By contrast, substituted derivatives of the holo homodimeric form of BOLA3 are formed and remain active toward cluster exchange.

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Reconstitution, characterization, and [2Fe–2S] cluster exchange reactivity of a holo human BOLA3 homodimer

Cited by 3 publications

References 56 publications

Cluster exchange reactivity of [2Fe‐2S]‐bridged heterodimeric BOLA1‐GLRX5

Cluster exchange reactivity of [2Fe‐2S]‐bridged heterodimeric BOLA1‐GLRX5

Spectroscopic and functional characterization of the [2Fe–2S] scaffold protein Nfu from Synechocystis PCC6803

Biochemical impact of a disease-causing Ile67Asn substitution on BOLA3 protein

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