Dioxygen controls the nitrosylation reactions of a protein-bound [4Fe4S] cluster.

Daniel B Grabarczyk,William K Myers,Philip A Ash,Kylie A Vincent,Erin L Dodd

doi:10.1039/c9dt00924h

Daniel B Grabarczyk, William K Myers + Show 3 more

Open Access

https://doi.org/10.1039/c9dt00924h

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Abstract

Iron-sulfur clusters are exceptionally tuneable protein cofactors, and as one of their many roles they are involved in biological responses to nitrosative stress. Both iron-sulfur proteins and synthetic model clusters are extremely sensitive to nitrosylation, tending towards rapid multi-step reaction and cluster degradation. Reaction of protein-bound iron-sulfur clusters with nitric oxide can be stopped at partial nitrosylation in vivo, and repair of protein-bound nitrosylated clusters is possible in the cellular environment. We have used a combination of infrared, EPR, and UV-visible spectroscopies to show that a model [4Fe4S] cluster-containing protein, A. ferroxidans high potential iron-sulfur protein (HiPIP), reacts with NO to give a product mixture dominated by Roussin's Black Salt (RBS) and Roussin's Red Ester (RRE) species. We have shown that O2 plays a critical role in controlling the major product of nitrosylation, with RBS-like products favoured under strictly anaerobic conditions and RRE favoured in the presence of trace O2. Moreover, addition of trace O2 to anaerobically nitrosylated samples induces conversion of RBS-like products to RRE. These findings may have implications for mechanisms of iron-sulfur cluster repair following nitrosative stress, suggest a crucial role for trace O2, and provide an important link between nitrosylation chemistry of iron-sulfur proteins and the well-established reactivity of synthetic iron-sulfur clusters.

Highlights

Bacteria are exposed to the free-radical gas nitric oxide (NO) in a wide variety of situations, for example during the mammalian immune response[1] and as an intermediate in denitrification.[2]
The [4Fe4S] cluster is bound to the protein via four cysteine ligands and the protein contains an internal disulfide bond involving two other cysteine ligands, one of which is directly adjacent to a cluster-binding cysteine.[37]
The identity of the protein was further verified by cyclic voltammetry, which showed a reversible redox process centred at +0.52 V vs. SHE (Fig. S2†) consistent with the potential reported by Bruscella et al.[38] for untagged A. ferroxidans high potential iron–sulfur protein (HiPIP)

Summary

Introduction

Bacteria are exposed to the free-radical gas nitric oxide (NO) in a wide variety of situations, for example during the mammalian immune response[1] and as an intermediate in denitrification.[2].

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