<scp> H ₂ </scp> ‐dependent modulation of tetrahydromethanopterin S‐methyltransferase (Mtr complex) activity by the small protein <scp>MtrR</scp> in <i>Methanosarcina mazei</i>

Small open reading frame (sORF)-encoded proteins, with less than 100 amino acids, have attracted increasing attention over the past decade after being overlooked due to limitations in classical methodologies. For the mesophilic archaeal model system Methanosarcina mazei, a high number of previously unannotated sORFs have recently been identified. However, the physiological role of most of the respective small proteins remains unknown. Here, we report on the functional characterization of the small ORF16-encoded small protein MtrR (49 amino acids). We demonstrate that MtrR forms oligomers localized at the cytoplasmic membrane. There, it interacts with the tetrahydrosarcinapterin S-methyltransferase (Mtr), a key membrane-bound complex of energy metabolism, and impacts its activity. In vitro interaction and in vivo copurification assays showed MtrR interaction with the Mtr complex, which was further validated by microscale thermophoresis analysis demonstrating a specific interaction with the MtrA subunit. Analyzing growth under varying molecular hydrogen (H₂) availability demonstrated that the mtrR deletion mutant showed significantly impaired growth in the presence of H₂, independent of the carbon source. Further, we observed induction of mtrR transcription in the presence of H₂. Consequently, we propose that MtrR fine-tunes the activity of the Mtr complex in response to fluctuating H₂ availabilities, allowing adaptation of the energy metabolism to changing environmental H₂ conditions.