Mechanisms of iron- and O2-sensing by the [4Fe-4S] cluster of the global iron regulator RirA.

Ma Teresa Pellicer Martinez,Jason C Crack,Melissa Yy Stewart,Jonathan D Todd,Nick E Le Brun,Andrew Wb Johnston,Dimitri A Svistunenko,Justin M Bradley,Myles R Cheesman

doi:10.7554/elife.47804

Ma Teresa Pellicer Martinez, Jason C Crack + Show 7 more

Open Access

https://doi.org/10.7554/elife.47804

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Journal: eLife	Publication Date: Sep 17, 2019
Citations: 29	License type: CC BY 4.0

Affiliation: University of East Anglia, University of Essex

Abstract

RirA is a global regulator of iron homeostasis in Rhizobium and related α-proteobacteria. In its [4Fe-4S] cluster-bound form it represses iron uptake by binding to IRO Box sequences upstream of RirA-regulated genes. Under low iron and/or aerobic conditions, [4Fe-4S] RirA undergoes cluster conversion/degradation to apo-RirA, which can no longer bind IRO Box sequences. Here, we apply time-resolved mass spectrometry and electron paramagnetic resonance spectroscopy to determine how the RirA cluster senses iron and O2. The data indicate that the key iron-sensing step is the O2-independent, reversible dissociation of Fe2+ from [4Fe-4S]2+ to form [3Fe-4S]0. The dissociation constant for this process was determined as Kd = ~3 µM, which is consistent with the sensing of 'free' iron in the cytoplasm. O2-sensing occurs through enhanced cluster degradation under aerobic conditions, via O2-mediated oxidation of the [3Fe-4S]0 intermediate to form [3Fe-4S]1+. This work provides a detailed mechanistic/functional view of an iron-responsive regulator.

Highlights

Iron–sulphur clusters are ubiquitous protein cofactors that play essential roles across all of life in processes as diverse as respiration, photosynthesis, and DNA replication (Beinert et al, 1997; Johnson et al, 2005), with recent evidence that they may be even more abundant than initially thought (Rouault, 2015)
In many bacteria, including such taxonomically diverse model organisms as Escherichia coli and Bacillus subtilis, iron uptake is under the control of the global iron regulator Fur (Ferric uptake regulator)
Pellicer Martinez, Crack, Stewart et al used a technique known as time-resolved mass spectrometry to examine the sensory response of the iron-sulphur cluster of RirA when different levels of iron were available

Summary

Introduction

Iron–sulphur clusters are ubiquitous protein cofactors that play essential roles across all of life in processes as diverse as respiration, photosynthesis, and DNA replication (Beinert et al, 1997; Johnson et al, 2005), with recent evidence that they may be even more abundant than initially thought (Rouault, 2015). Elucidating the precise nature of their roles is a major challenge, which is often complicated by the extreme reactivity of the cluster to O2 and other gases This very sensitivity has been exploited through the evolution of iron–sulphur cluster-containing transcriptional regulators that enable cells to sense and respond to, for example, oxidative stress and changes in concentrations of metabolically important species such as O2 and iron (Beinert and Kiley, 1999; Crack et al, 2014a). Iron is sensed through the availability and binding of Fe2+ directly to the Fur protein, resulting in a Pellicer Martinez et al eLife 2019;8:e47804. Pellicer Martinez, Crack, Stewart et al used a technique known as time-resolved mass spectrometry to examine the sensory response of the iron-sulphur cluster of RirA when different levels of iron were available. The data provide a comprehensive mechanistic and functional view of an important iron-responsive regulator

Results and discussion

Conclusions

Materials and methods

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