Spatial and temporal control of mitochondrial H ₂ O ₂ release in intact human cells

Abstract

Hydrogen peroxide (H2O2) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2O2. Here, we employed a genetically encoded high‐affinity H2O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria‐released H2O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously observed differences between cell types. Our data suggest that H2O2‐mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.

NADPH and Mitochondrial Quality Control as Targets for a Circadian-Based Fasting and Exercise Therapy for the Treatment of Parkinson’s Disease

“…It also suggests that dysfunctional MQC is associated with a heavier load of other ROS/RNS, such as H 2 O 2 , which is synthesized from O 2 •− by mitochondrial SOD2. H 2 O 2 could also potentially be used to identify damaged mitochondria [ 213 ]. Although both O 2 •− and H 2 O 2 can stimulate autophagy, SOD2 overexpression, which increases H 2 O 2 levels, decreased autophagy, while SOD2 knockdown, which increases O 2 •− levels, increased autophagy [ 31 ].…”

Maintenance of small molecule redox homeostasis in mitochondria

“…Since ROS are mainly generated within the cristae space where the majority of the respiratory chain is localised, diffusion of, for example H 2 O 2 across the IMM towards the matrix might be kinetically favoured over diffusion through cristae junctions, peripheral IMS and across the OMM. Although this has not been addressed directly, studies using genetically encoded H 2 O 2 sensors and antimycin A treatment that induced superoxide anion release towards the IMS demonstrated an increase in matrix H 2 O 2 levels [42,43]. Conversely, proteomic experiments investigating the location of redoxmodified proteins upon treatment with complex I or complex III inhibitors showed thatas expected from the directionality of H 2 O 2 releasemainly proteins in matrix and IMS were modified, respectively [75].…”

“…It thus remains unclear whether H 2 O 2 is efficiently reduced to water in the IMS. On the other hand, it might be particularly this absence of H 2 O 2 reductive enzymes in the IMS that allows mitochondrial H 2 O 2 release and signalling in the first place [42,88]. This is indicated by the fact that artificial targeting of a peroxiredoxin to the IMS attenuates mitochondrial H 2 O 2 signalling [89].…”

“…This allows the prolonged interaction with and the transfer of single electrons onto molecular oxygen leading to the generation of superoxide anions [41]. This becomes especially prominent in cells grown in the presence of nutrients which require for their catabolism electron flux through the respiratory chain [42,43]. A different example are cells that rely on increased de novo pyrimidine biosynthesis and thus an increased turnover of dihydroorotate to orotate by DHO-DH [44].…”

“…pyruvate dehydrogenase, α‐ketoglutarate dehydrogenase) [106–111]. As discussed above also diffusion of H 2 O 2 from the IMS might contribute to the H 2 O 2 load in the matrix [42].…”