Zinc(II) binding on human wild-type ISCU and Met140 variants modulates NFS1 desulfurase activity

Nicholas G Fox,Alain Martelli,Joseph F Nabhan,Jay Janz,Oktawia Borkowska,Christine Bulawa,Wyatt W Yue

doi:10.1016/j.biochi.2018.07.012

Nicholas G Fox, Alain Martelli + Show 5 more

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https://doi.org/10.1016/j.biochi.2018.07.012

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Abstract

Human de novo iron-sulfur (Fe-S) assembly complex consists of cysteine desulfurase NFS1, accessory protein ISD11, acyl carrier protein ACP, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN binds the NFS1-ISD11-ACP-ISCU complex (SDAU), to activate the desulfurase activity and Fe-S cluster biosynthesis. In the absence of FXN, the NFS1-ISD11-ACP (SDA) complex was reportedly inhibited by binding of recombinant ISCU. Recent studies also reported a substitution at position Met141 on the yeast ISCU orthologue Isu, to Ile, Leu, Val, or Cys, could bypass the requirement of FXN for Fe-S cluster biosynthesis and cell viability. Here, we show that recombinant human ISCU binds zinc(II) ion, as previously demonstrated with the E. coli orthologue IscU. Surprisingly, the relative proportion between zinc-bound and zinc-depleted forms varies among purification batches. Importantly the presence of zinc in ISCU impacts SDAU desulfurase activity. Indeed, removal of zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SDA alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SDA. Taking into consideration the inhibition of desulfurase activity by zinc-bound ISCU, we characterized wild type ISCU and the M140I, M140L, and M140V variants under both zinc-bound and zinc-depleted conditions, and did not observe significant differences in the biochemical and biophysical properties between wild-type and variants. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SDA complex. This study therefore identifies an important regulatory role for zinc-bound ISCU in modulation of the human Fe-S assembly system in vitro and reports no ‘FXN bypass’ effect on mutations at position Met140 in human ISCU. Furthermore, this study also calls for caution in interpreting studies involving recombinant ISCU by taking into consideration the influence of the bound zinc(II) ion on SDAU complex activity.

Highlights

Iron-sulfur (Fe-S) clusters are prosthetic groups required for critical cellular functions including oxidative respiration, DNA repair, and the biosynthesis of other cofactors.[1, 2] The iron sulfur (Fe-S) biosynthetic pathway in humans is located in the mitochondrial matrix and is initiated by a protein complex of NFS1, ISD11, ISCU and frataxin (FXN) (‘protein complex composed of NFS1 (SDUF)’).[3,4,5,6] Recently, this protein complex was shown to be associated with an additional component, the acyl carrier protein (ACP, known in human as NDUFAB1),[7, 8] whether acyl carrier transfer protein (ACP) is an intrinsic component of the complex in vivo remains to be determined
E. coli IscU was previously shown to exist in two interconvertible conformations, comprising a structured and a disordered form that can be influenced by the zinc loading status.[21, 24, 25]
The metalconferred thermostability was reflected by far-UV circular dichroism spectra consistent with zincbound ISCU displaying a higher helical percentage (28% compared to 20%) and less disordered percentage (35% compared to 41%) than the zinc-depleted ISCU (Figure 1C,D)

Summary

Introduction

Iron-sulfur (Fe-S) clusters are prosthetic groups required for critical cellular functions including oxidative respiration, DNA repair, and the biosynthesis of other cofactors.[1, 2] The Fe-S biosynthetic pathway in humans is located in the mitochondrial matrix and is initiated by a protein complex of NFS1, ISD11, ISCU and frataxin (FXN) (‘SDUF’).[3,4,5,6] Recently, this protein complex was shown to be associated with an additional component, the acyl carrier protein (ACP, known in human as NDUFAB1),[7, 8] whether ACP is an intrinsic component of the complex in vivo remains to be determined. The cysteine desulfurase NFS1 (homolog of bacterial IscS or yeast Nfs1) catalyzes the pyridoxal phosphate (PLP)dependent conversion of L-cysteine to L-alanine, and generates a persulfide species that delivers the sulfane sulfur to the Fe-S scaffold protein, ISCU (homolog of yeast Isu or bacterial IscU).[9, 10] ISD11 (LYRM4) is a eukaryotic-specific protein belonging to the Leu-Tyr-Arg (LYR) superfamily of small and basic proteins, which interacts, stabilizes, and may regulate NFS1 activity.[7, 11,12,13,14,15,16]. Frataxin (FXN, homolog of yeast Yfh[1] or E. coli CyaY) is an allosteric regulator of the Fe-S assembly complex, with human FXN shown to stimulate the rate of cysteine desulfurase activity[17] and iron sulfur cluster biosynthesis[18] in vitro, whereas the bacterial homolog CyaY appears to inhibit the counterpart desulfurase IscS.[19]

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