Biology subjects

Svistunenko, D. A.

Publications and source records attributed to Svistunenko, D. A..

3 recordsLinked to original sources

bioRxiv2025

Human mitochondrial ferritin exhibits highly unusual iron-O2 chemistry distinct from that of cytosolic ferritins

Ferritins are ubiquitous proteins that function in iron storage and detoxification. Mammalian tissues that are metabolically highly active contain, in addition to the ubiquitous cytosolic ferritin, a ferritin that is localised to mitochondria. Mitochondrial ferritin (FtMt) functions to protect against oxidative stress and is found at higher levels in disease states that are associated with abnormal iron accumulation, such as Alzheimers and Parkinsons. Here we demonstrate that, despite 80% sequence identity with cytosolic human H-chain ferritin, Fe2+ oxidation at the catalytic diiron ferroxidase centre of FtMt proceeds via a distinct mechanism involving radical formation on a strictly conserved Tyr residue (Tyr34), and a mixed-valent ferroxidase centre (MVFC) that is readily detected under the O2-limiting conditions typical of mitochondria. Tyr34 is key for the activation of O2 and stability of the MVFC. The highly unusual iron-O2 chemistry exhibited by FtMt demonstrates that high levels of sequence identity between enzymes does not guarantee similarity of catalytic mechanism; here we explore the possible origin of the mechanistic differences between FtMt and cytosolic human H-chain ferritin.

biochemistry↗

bioRxiv2024

Iron and redox sensing in plants: properties of the hemerythrin-like domains of Arabidopsis BRUTUS and BRUTUS-LIKE2 proteins

Iron uptake in plants is negatively regulated by highly conserved hemerythrin (Hr) E3 ubiquitin ligases exemplified by Arabidopsis thaliana BRUTUS (BTS). Physiological studies suggest these are the elusive plant iron sensors, but biochemical evidence is lacking. Here we demonstrate that the N-terminal domains of BTS and BTS-LIKE2 (BTSL2) respectively bind three and two diiron centres within three closely packed Hr-like subdomains. The centres can be reversibly oxidized by O2 and H2O2, resulting in a di-Fe3+ form that is non-labile. In the reduced state, a proportion of the iron becomes labile, based on accessibility to Fe2+ chelators and reconstitution experiments, consistent with dynamic iron binding. Impaired iron binding and altered redox properties in the BTS dgl variant correlate with diminished capacity to suppress the downstream signalling cascade. These data provide the biochemical foundation for a mechanistic model of how BTS/Ls function as iron sensors that are unique to the plant kingdom.

biochemistry↗

bioRxiv2021

Rigidifying a de novo enzyme increases activity and induces a negative activation heat capacity

Conformational sampling profoundly impacts the overall activity and temperature dependence of enzymes. Peroxidases have emerged as versatile platforms for high value biocatalysis owing to their broad palette of potential biotransformations. Here, we explore the role of conformational sampling in mediating a de novo peroxidases activity. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending towards a more globally rigid structure. This is correlated with increases both stability and activity. Notably, these effects are concomitant with the emergence of curvature in the temperature-activity profile, trading off activity gains at ambient temperature with losses at high temperatures. We apply macromolecular rate theory (MMRT) to understand enzyme temperature dependence data. These data point to an increase in protein rigidity associated with a difference in the distribution of protein dynamics between the ground and transition state. We compare the thermodynamics of the de novo enzyme activity to those of a natural peroxidase, horseradish peroxidase. We find that the native enzyme resembles the rigidified de novo enzyme in terms of the thermodynamics of enzyme catalysis and the putative distribution of protein dynamics between the ground and transition state. The addition of TFE apparently causes C45 to behave more like the natural enzyme. Our data suggest robust, generic strategies for improving biocatalytic activity by manipulating protein rigidity; for functional de novo protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign or directed evolution.

biochemistry↗