Of the reactive nitrogen species, the chemical biology of nitroxyl is one of the least understood. In part, this is due to the transient nature of HNO which requires use of donors such as Angeli’s salt for its study. In mammals, HNO shows overlapping bioactivity with nitric oxide despite their distinct fundamental chemistry. Thiols like glutathione and cysteine are readily oxidized by nitroxyl (forming disulfides) and have mitigating effects on its bioactivity. In recent years, other LMW thiols such as bacillithiol (BSH) have emerged in low GC Gram-positive bacteria and represent an overlooked area of research.
BSH was synthesized following work developed by Armstrong & Sulikowski and Hamilton. Pure BSH was obtained in amounts of 150-300 mg per batch (3-4% over 10 steps) and was used to explore its chemistry with HNO. Equimolar and excess Angeli’s Salt and 2-bromo Piloty’s acid were incubated with bacillithiol, glutathione, cysteine, N-acetylcysteine, and glucosamine-N-cysteine and monitored by mass spectrometry. These reactions led to the formation of disulfides as the major products, except for BSH which favored the formation of the sulfinamide adduct. The total thiol content of bacillithiol/HNO reactions was assessed using a well-known derivatization assay with fluorescence coupled high performance liquid chromatography. On average, 3% of the theoretical bacillithiol concentration was recovered compared to 18% for glutathione, leaving a significant amount of thiol unaccounted. MS analysis led to the discovery of an unknown product in bacillithiol and glutathione incubations, believed to be a cyclic-sulfinamide whose molecular formula was confirmed by high resolution mass spectrometry. Formation of the open chain and cyclic sulfinamides in bacillithiol is thought to be driven by the malate group of its structure, since the synthetic analog glucosamine-N-cysteine (treated with HNO) gives disulfide and negligible sulfinamide.
Cell studies used Bacillus subtilis CU1065 (wild-type) and HB110079 (unable to produce bacillithiol) strains which were grown in rich and minimal media and treated with HNO during exponential growth. HNO proved to be toxic to both cell strains but more severely to HB110079, showing that bacillithiol imparts protection against HNO stress. Bacillithiol from CU1065 cells in minimal medium was quantified by fluorescence coupled HPLC after HNO treatment and showed a decrease in the total bacillithiol pool compared to controls. BSH levels could not be recovered by 1,4-dithiothreitol (DTT) in accordance with sulfinamide chemistry, suggesting the formation of bacillithiol-sulfinamide and likely cyclic bacillithiol sulfinamide.
Collectively this work shows that bacillithiol imparts protection against HNO stress to Bacillus subtilis, and that HNO and bacillithiol result in a distinct product profile not seen in other low molecular weight thiols, which prefers sulfinamides over disulfides. Moreover, HNO depletes the BSH pool to a measurable degree in Bacillus subtilis and BSH cannot be recovered by DTT, suggesting that bacillithiol sulfinamide likely forms in-vivo in line with the reaction profile observed during in vitro studies.