Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.

Mismatch repair (MMR) deficiencies are a hallmark of various cancers causing accumulation of DNA mutations and mismatches, which often results in chemotherapy resistance. Metalloinsertor complexes, including [Rh(chrysi)(phen)(PPO)]Cl2 (Rh-PPO), specifically target DNA mismatches and selectively induce cytotoxicity within MMR-deficient cells. Here, we present an in vivo analysis of Rh-PPO, our most potent metalloinsertor. Studies with HCT116 xenograft tumors revealed a 25% reduction in tumor volume and 12% increase in survival with metalloinsertor treatment (1 mg/kg; nine intraperitoneal doses over 20 d). When compared to oxaliplatin, Rh-PPO displays ninefold higher potency at tumor sites. Pharmacokinetic studies revealed rapid absorption of Rh-PPO in plasma with notable accumulation in the liver compared to tumors. Additionally, intratumoral metalloinsertor administration resulted in enhanced anticancer effects, pointing to a need for more selective delivery methods. Overall, these data show that Rh-PPO inhibits xenograft tumor growth, supporting the strategy of using Rh-PPO as a chemotherapeutic targeted to MMR-deficient cancers.

Nitrogenase is the sole enzyme responsible for the ATP‐dependent conversion of atmospheric dinitrogen into the bioavailable form of ammonia (NH3), making this protein essential for the maintenance of the nitrogen cycle and thus life itself. Despite the widespread use of the Haber–Bosch process to industrially produce NH3, biological nitrogen fixation still accounts for half of the bioavailable nitrogen on Earth. An important feature of nitrogenase is that it operates under physiological conditions, where the equilibrium strongly favours ammonia production. This biological, multielectron reduction is a complex catalytic reaction that has perplexed scientists for decades. In this review, we explore the current understanding of the molybdenum nitrogenase system based on experimental and computational research, as well as the limitations of the crystallographic, spectroscopic, and computational techniques employed. Finally, essential outstanding questions regarding the nitrogenase system will be highlighted alongside suggestions for future experimental and computational work to elucidate this essential yet elusive process.

Up to twenty percent of solid tumors are characterized by DNA mismatch repair (MMR) deficiency and microsatellite instability (MSI) that confer resistance to standard of care chemotherapy. MMR-deficient cancers have an increased mutation rate and accumulate DNA mismatches. We previously described a class of compounds, rhodium metalloinsertors, that bind DNA mismatches with high specificity and selectivity and have potential as targeted therapy. [Rh(chrysi)(phen)(PPO)] 2+ (RhPPO) is the most potent, selective compound in this class and acts by targeting DNA mismatches, resulting in preferential cytotoxicity to MMR-deficient cancers. To explore further the cellular mechanism of action of RhPPO, we conjugated the metal complex to a fluorescent probe, cyanine 3 (Cy3). RhPPO-Cy3 binds DNA mismatches and retains the selectivity and potent cytotoxic activity of RhPPO for MMR-deficient cell lines. RhPPO-Cy3 forms discrete foci in the cell nucleus that overlap with sites of DNA damage, suggesting that the lesions occur at or near DNA mismatch sites. RhPPO-Cy3 foci persist over time, despite initial processing of the lesion and recruitment of repair proteins, consistent with the idea that the complex binding to a mismatch prevents repair. RhPPO-Cy3 binding does not lead to activation of p53 and the apoptotic pathway. Together, these findings support the idea that RhPPO-Cy3 binding leads to irreversible DNA damage at DNA mismatches that enables selective cytotoxicity to MMR-deficient cells.