Nonsymmetrical oxygen-bridged binuclear copper centers have been proposed and modeled as intermediates and transition states in several C─H oxidation pathways, leading to the postulation that structural dissymmetry enhances the reactivity of the bridging oxygen. However, experimentally characterizing the structure and reactivity of these transient species is remarkably challenging. Here, we report the high-pressure synthesis of a metastable nonsymmetrical dicopper-μ-oxo compound with exceptional reactivity toward the mono-oxygenation of aliphatic C─H bonds. The nonequivalent coordination environment of copper stabilizes localized mixed valency and greatly enhances the hydrogen atom abstraction activity of the bridging oxygen, enabling room-temperature hydroxylation of methane under pressure. These findings highlight the role of dissymmetry in the reactivity of binuclear copper centers and demonstrate precise control of molecular structures by mechanical means.

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs ( c and c -2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c -2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c ₁ for inducible knockdown. Translational repression of cyt c and cyt c ₁ was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c -2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c -2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c -2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution. SIGNIFICANCE STATEMENT: Mitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion of Plasmodium malaria parasites is a major drug target that has many differences from human cells and remains poorly studied. One key difference from humans is that malaria parasites express two cytochrome c proteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cyt c is essential for ETC function and parasite viability while the second, more divergent protein has unusual structural and biochemical properties and is not required for growth of blood-stage parasites. This work elucidates key biochemical properties and evolutionary differences in the mitochondrial ETC of malaria parasites.

Although covalently crosslinked gelatin hydrogels have been investigated for use in 3D cell culture due to inherent bioactivity and proliferation within the denatured collagen precursor, the stability of the matrix, and relatively inexpensive synthesis, current systems lack precise control over mechanical properties, including homogeneity, stiffness, and efficient diffusion of nutrients to embedded cells. Difficulties in modifying gel matrix composition and functionalization have limited the use of covalently crosslinked gelatin hydrogels as a three-dimensional (3D) cell culture medium, lacking the ability to tailor the microenvironment for specific cell types. In addition, the currently utilized chain-growth photopolymerization mechanism for crosslinking hydrogels has a potential for side reactions between the matrix backbone and components of the cell surface, requires a high concentration of radicals for initiation, and only cures with long irradiation times, which could lead to cytotoxicity. To overcome these limitations, a superfast curing reaction mechanism, in which a thiol monomer reacts efficiently with non-homopolymerizable alkenes, is suggested. This mechanism reliably produces a well-defined matrix that does not require a high radical concentration for photoinitiation. Mechanical customization of the hydrogel is largely achievable through variation in degree of functionalization of the gelatin backbone, dependent on reaction conditions such as pH, allyl concentration, and time. This work provides a mechanistic framework for GelAGE hydrogel fabrication by elucidating the molecular mechanism of gelatin functionalization with AGE and the thiol-ene crosslinking reactions controlling network stiffness. These insights provide the foundation for engineering hydrogels that mimic the viscoelastic and structural characteristics of cartilage, enabling advanced in vitro models for osteoarthritis research.