Roles of Met and Cys Residues of ROS/RCS sensor HprS in Bacterial Periplasmic Space

Reactive oxygen species (ROS), including superoxide, hydrogen peroxide, hydroxy radical, and singlet oxygen, can cause oxidative damage to cellular components, including proteins. Amino acid residues rich in sulfur, such as methionine (Met) and cysteine (Cys), are particularly susceptible to oxidation by ROS due to the sensitivity of sulfur atoms to oxidizing substances.

Met can be oxidized to methionine sulfoxide by ROS, particularly hydrogen peroxide. On the other hand, hypochlorous acid, a reactive chlorine species (RCS), can react with methionine to form an unstable intermediate that eventually leads to methionine sulfoxide formation. Cysteine sulfenic acid, another oxidized form of Cys, is highly reactive and can form disulfide bonds within the same protein or with thiol groups of other proteins. Disulfide bond formation affects protein structure and function and has been shown to regulate the activity of enzymes.

ROS and RCS also play a crucial role in the defense against pathogenic bacteria in animals. Neutrophils utilize NADPH oxidase and superoxide dismutase enzymes to produce hydrogen peroxide and superoxide, respectively. Hydrogen peroxide is converted to hypochlorous acid through a myeloperoxidase-mediated reaction with chloride ions. These reactive species are involved in bacterial cell invasion and the destruction of essential proteins necessary for bacterial growth.

Gram-negative bacteria such as Escherichia coli, have evolved mechanisms to counteract ROS and RCS. They produce enzymes like catalase, NADH peroxidase, and superoxide dismutase to eliminate intracellular ROS. Thioredoxin and thioredoxin reductase reduce oxidized cysteine residues, and the transcriptional regulator OxyR controls the expression of ROS-scavenging enzymes and oxidized protein repair enzymes. Similarly, the periplasmic region of bacteria contains the cysteine sulfenic acid reduction system involving DsbC, DsbD, and DsbG to repair oxidized proteins.

The HClO-responsive transcription factor HypT acts as an intracellular HClO sensor. It is part of the LysR-type transcriptional regulator family and undergoes activation upon oxidation of specific methionine residues. HypT regulates the expression of genes involved in iron acquisition, methionine, and cysteine biosynthesis.

The two-component regulatory system (TCS) consisting of the sensor kinase HprS and the response regulator HprR is responsible for extracellular ROS sensing. Through crosstalk with the copper response TCS, HprS/R TCS controls the expression of genes involved in phosphate relay. HprS has specific methionine residues that are critical for its activation and response to oxidative stress.

In a new study published in the peer reviewed Journal FEBS Letters by Kotaro Yamaji, Rumine Taniguchi, Hiroyuki Urano and led by Associate Professor Hiroshi Ogasawara at Shinshu University in Japan, they investigated the mechanism underlying the oxidative stress response mediated by HprS in the bacterial periplasmic space. The study aimed to understand the roles of individual methionine and cysteine residues in HprS in response to hypochlorite (HClO) in the external environment.

The authors evaluated the HprS/R target gene hiuH promoter activity using various HprS variants with alanine substitutions of specific methionine residues. They focused on Met72, Met73, Met153, and Met225 and analyzed their induction of hiuH expression in the presence and absence of HClO. Moreover, the authors used structural predictions generated by Alphafold to gain insights into the potential impact of these substitutions on protein structure and activation. These data suggested the Met72 and Met73 are important sites that regulate the activity of HprS by interacting with other proteins. Furthermore, They also focused on the role of cysteine residues, particularly Cys165 and Cys22, in HprS homolog. They analyzed the induction of hiuH–lacZ expression in the presence of HClO using these substitutions mutants and compared the differences between E. coli HprS (G22/C165) and HprS homolog in the close relatives of E. coli (C22/S165). As a results, it is suggested that one of the amino acids residues G22 or C165 adjacent to each other in the transmembrane domain is Cys, which is important for the activation of the HprS homolog. In addition, structural predictions were suggested the potential complex formation with other factor via disulfide bonds due to the Cys165.

In conclusion, they provided valuable insights into their roles of ROS/RCS sensor in the bacterial oxidative stress response in the periplasmic space, furthering our understanding of the intricate mechanisms involved in cellular defense against oxidative damage.