Adaptation of eukaryotic cells to oxygen-poor environments has driven metabolic changes in mitochondria, notably shifting from oxygen-dependent to anaerobic energy metabolism. However, how the mitochondrial protein import machinery adapts in anaerobes remains poorly understood, although oxygen is crucial for this process, particularly for oxidative folding of small Tim (sTim) chaperones. sTim heterohexameric complexes guide imported proteins within the mitochondrial intermembrane space (IMS). Their function depends on conserved twin cysteines, oxidized by the mitochondrial import and assembly (MIA) pathway to stabilize their structure via disulfide bridges. The folding requires molecular oxygen or cytochrome c as electron acceptors, linking sTim folding to respiration. This study elucidates how the sTim/MIA pathway is reshaped in anaerobic types of mitochondria such as hydrogenosomes. Through structural and homology analyses across anaerobic eukaryotes, three modifications of the sTim/MIA system were identified: (i) a disulfide relay-independent system with sTims lacking twin cysteines (sTim-cys), (ii) absence of sTim/MIA components, and (iii) a conventional sTim/MIA system linked to fumarate reduction. The sTim-cys system found in Metamonada was studied in Trichomonas vaginalis hydrogenosomes. Structural modeling, in vitro, and in situ analyses revealed that despite lacking canonical cysteines, sTim-cys proteins maintain the helix-loop-helix architecture with the central loop involved in targeting to the IMS, and assemble into complexes stabilized by electrostatic interactions. Single-particle analysis confirmed their six-fold symmetry, similar to conventional sTim heterohexamers. These findings provide insights into the evolutionary shaping of sTim/MIA pathways in anoxic environments, contributing to our understanding of mitochondrial biogenesis across diverse eukaryotes.

Adaptation of eukaryotic cells to oxygen-poor environments has driven metabolic changes in mitochondria, notably shifting from oxygen-dependent to anaerobic energy metabolism. However, how the mitochondrial protein import machinery adapts in anaerobes remains poorly understood, although oxygen is crucial for this process, particularly for oxidative folding of small Tim (sTim) chaperones. sTim heterohexameric complexes guide imported proteins within the mitochondrial intermembrane space (IMS). Their function depends on conserved twin cysteines, oxidized by the mitochondrial import and assembly (MIA) pathway to stabilize their structure via disulfide bridges. The folding requires molecular oxygen or cytochrome c as electron acceptors, linking sTim folding to respiration. This study elucidates how the sTim/MIA pathway is reshaped in anaerobic types of mitochondria such as hydrogenosomes. Through structural and homology analyses across anaerobic eukaryotes, three modifications of the sTim/MIA system were identified: (i) a disulfide relay-independent system with sTims lacking twin cysteines (sTim-cys), (ii) absence of sTim/MIA components, and (iii) a conventional sTim/MIA system linked to fumarate reduction. The sTim-cys system found in Metamonada was studied in Trichomonas vaginalis hydrogenosomes. Structural modeling, in vitro, and in situ analyses revealed that despite lacking canonical cysteines, sTim-cys proteins maintain the helix-loop-helix architecture with the central loop involved in targeting to the IMS, and assemble into complexes stabilized by electrostatic interactions. Single-particle analysis confirmed their six-fold symmetry, similar to conventional sTim heterohexamers. These findings provide insights into the evolutionary shaping of sTim/MIA pathways in anoxic environments, contributing to our understanding of mitochondrial biogenesis across diverse eukaryotes.