A Red Fluorescent Protein-Based Probe for Detection of Intracellular Reactive Sulfane Sulfur

Abstract

Reactive sulfane sulfur, including persulfide and polysulfide, is a type of regular cellular component, playing an antioxidant role. Its function may be organelle-dependent; however, the shortage of probes for detecting organellar reactive sulfane sulfur has hindered further investigation. Herein, we reported a red fluorescent protein (mCherry)-based probe for specifically detecting intracellular reactive sulfane sulfur. By mutating two amino acid residues of mCherry (A150 and S151) to cysteine residues, we constructed a mCherry mutant, which reacted with reactive sulfane sulfur to form an intramolecular –Sn– bond (n ≥ 3). The bond largely decreased the intensity of 610 nm emission (excitation at 587 nm) and slightly increased the intensity of 466 nm emission (excitation at 406 nm). The 466/610 nm emission ratio was used to indicate the relative abundance of reactive sulfane sulfur. We then expressed this mutant in the cytoplasm and mitochondria of Saccharomyces cerevisiae. The 466/610 nm emission ratio revealed that mitochondria had a higher level of reactive sulfane sulfur than cytoplasm. Thus, the mCherry mutant can be used as a specific probe for detecting reactive sulfane sulfur in vivo.

A cascade of sulfur transferases delivers sulfur to the sulfur‐oxidizing heterodisulfide reductase‐like complex

“…Hm. denitrificans possesses two candidate transporters (SoxT1A and SoxT1B, Figure 1) encoded in close proximity to the genes encoding Sox proteins that are involved in the initial steps of thiosulfate oxidation and the genes for the cytoplasmic sHdr system (Li, Koch, et al, 2023;Li, Törkel, et al, 2023). However, evidence supporting the proposed sulfur transport has yet to be presented.…”

“…The polypeptides sHdrA and sHdrC2 as well as LbpA2 (aq_402) were identified, but were also present in the control sample (Table S4). Of the remaining proteins, 85 were associated with protein biosynthesis and degradation, and 24 were related to known or proposed functions of TusA other than sulfur oxidation, such as tRNA modification, molybdopterin cofactor biosynthesis and regulation (Dahl et al, 2013;Ikeuchi et al, 2006;Li, Törkel, et al, 2023). To gain further insight into the interaction partners of AqTusA, the re-purified samples were applied to SDS-PAGE (Figure 8a).…”

Sulfane Sulfur Is a Strong Inducer of the Multiple Antibiotic Resistance Regulator MarR in Escherichia coli

“…These modifications may be induced specifically by sulfane sulfur, as other oxidative reagents, such as H 2 O 2 , organic peroxides, and disulfide, cannot induce the formation of multiple sulfur links between two Cys residues. This concept has been applied to design sulfane sulfur-specific fluorescence proteins which, due to the space restriction of the Cys residues involved, cannot form disulfide links, but can form trisulfide and tetrasulfide links [ 45 , 46 ]. Disulfide formation can be achieved by the induction of sulfane sulfur as well as H 2 O 2 and other oxidative reagents.…”

Detection of sulfane sulfur species in biological systems

“…In 2019, Liu et al reported two protein-based fluorescent probes for sulfane sulfurs: one based on green fluorescent protein (GFP) and the other based on red fluorescent protein (mCherry) [ 33 ]. They first carried out 3D structure analyses on the proteins and identified possible sites for mutation.…”