Nitric oxide (NO) is a key signaling molecule involved in numerous physiological and pathological processes within the human body. This review specifically examines the involvement of NO in age-related diseases, focusing on the cardiovascular, nervous, and immune systems. The discussion delves into the mechanisms of NO signaling in these diseases, emphasizing the post-translational modifications of involved proteins, such as S-nitrosation and nitration. The review also covers the dual nature of NO, highlighting both its protective and harmful effects, determined by concentration, location, and timing. Additionally, potential therapies that modulate NO signaling, including the use of NO donors and nitric oxide synthases (NOSs) inhibitors in the treatment of cardiovascular, neurodegenerative, and oncological diseases, are analyzed. Particular attention is paid to the methods for the determination of NO and its derivatives in the context of illness diagnosis and monitoring. The review underscores the complexity and dual role of NO in maintaining cellular balance and suggests areas for future research in developing new therapeutic strategies.

Δ¹-Dehydrogenation of 3-ketosteroids catalyzed by flavin adenine dinucleotide (FAD)-dependent 3-ketosteroid dehydrogenases (Δ¹-KSTD) is a crucial step in steroid degradation and synthesis of several steroid drugs. The catalytic mechanism assumes the formation of a double bond in two steps, proton abstraction by tyrosyl ion, and a rate-limiting hydride transfer to FAD. This hypothesis was never verified by quantum-mechanical studies despite contradictory results from the kinetic isotope effect (KIE) reported in 1960 by Jerussi and Ringold [Biochemistry1965, 4 (10)]. In this paper, we present results that reconcile the mechanistic hypothesis with experimental evidence. Quantum mechanics/molecular mechanics molecular dynamics simulations show that the proposed mechanism is indeed the most probable, but barriers associated with substrate activation (13.4–16.3 kcal·mol^–1) and hydride transfer (15.5–18.0 kcal·mol^–1) are very close (1.7–2.1 kcal·mol^–1), which explains normal KIE values for steroids labeled either at C1 or C2 atoms. We confirm that tyrosyl ion acting as the catalytic base is indeed necessary for efficient activation of the steroid. We explain the lower value of the observed KIE (1.5–3.5) by the nature of the free energy surface, the presence of diffusion limitation, and to a smaller extent, conformational changes of the enzyme upon substrate binding. Finally, we confirm the Ping-Pong bi–bi kinetics of the whole Δ¹-dehydrogenation and demonstrate that substrate binding, steroid dehydrogenation, and enzyme reoxidation proceed at comparable rates.

According to the current paradigm, the metal–hydroxo bond in a six-coordinate porphyrin complex is believed to be significantly less reactive in ligand substitution than the analogous metal–aqua bond, due to a much higher strength of the former bond. Here, we report kinetic studies for nitric oxide (NO) binding to a heme-protein model, acetylated microperoxidase-11 (AcMP-11), that challenge this paradigm. In the studied pH range 7.4–12.6, ferric AcMP-11 exists in three acid–base forms, assigned in the literature as [(AcMP-11)Fe^III(H₂O)(HisH)] (1), [(AcMP-11)Fe^III(OH)(HisH)] (2), and [(AcMP-11)Fe^III(OH)(His^–)] (3). From the pH dependence of the second-order rate constant for NO binding (k_on), we determined individual rate constants characterizing forms 1–3, revealing only a ca. 10-fold decrease in the NO binding rate on going from 1 (k_on⁽¹⁾ = 3.8 × 10⁶ M^–1 s^–1) to 2 (k_on⁽²⁾ = 4.0 × 10⁵ M^–1 s^–1) and the inertness of 3. These findings lead to the abandonment of the dissociatively activated mechanism, in which the reaction rate can be directly correlated with the Fe–OH bond energy, as the mechanistic explanation for the process with regard to 2. The reactivity of 2 is accounted for through the existence of a tautomeric equilibrium between the major [(AcMP-11)Fe^III(OH)(HisH)] (2a) and minor [(AcMP-11)Fe^III(H₂O)(His^–)] (2b) species, of which the second one is assigned as the NO binding target due to its labile Fe–OH₂ bond. The proposed mechanism is further substantiated by quantum-chemical calculations, which confirmed both the significant labilization of the Fe–OH₂ bond in the [(AcMP-11)Fe^III(H₂O)(His^–)] tautomer and the feasibility of the tautomer formation, especially after introducing empirical corrections to the computed relative acidities of the H₂O and HisH ligands based on the experimental pK_a values. It is shown that the “effective lability” of the axial ligand (OH^–/H₂O) in 2 may be comparable to the lability of the H₂O ligand in 1.

This review covers highlights of the work performed in the van Eldik group on inorganic reaction mechanisms over the past two decades in the form of a personal journey. Topics that are covered include, from NO to HNO chemistry, peroxide activation in model porphyrin and enzymatic systems, the wonder-world of Ru^III(edta) chemistry, redox chemistry of Ru(III) complexes, Ru(II) polypyridyl complexes and their application, relevant physicochemical properties and reaction mechanisms in ionic liquids, and mechanistic insight from computational chemistry. In each of these sections, typical examples of mechanistic studies are presented in reference to related work reported in the literature.

The antimetastatic ruthenium(III) complex (H₂Im)[trans-RuCl₄(HIm)(DMSO)] (NAMI-A) as well as its two analogues (H₂Ind)[trans-RuCl₄(HInd)(DMSO)] (Ru-Ind) and (HIsq)[trans-RuCl₄(Isq)(DMSO)] (Ru-Isq) (HIm–imidazole, HInd–indazole, Isq–isoquinoline, DMSO–dimethyl sulfoxide) were tested for their effect on endothelial cell functions in vitro on human skin microvascular endothelial cells (HSkMEC) and human endothelial progenitor cells (HPEC-CB.2) under normoxic (21 % O₂) and hypoxic (1 % O₂) conditions. All studied complexes showed very low cytotoxicity profiles towards both mature microvascular and precursor endothelial cells (ECs), independently of oxygen concentration. Among tested compounds Ru-Ind exhibited the highest cytotoxicity. The antiangiogenic activity of ruthenium complexes was evaluated for their influence on pseudo-vessels formation by microvascular endothelial cells (HSkMEC) because of their involvement in melanoma progression. Our studies indicated that Ru-Ind and Ru-Isq exhibited hypoxia- and dose-dependent-inhibition of angiogenesis on Matrigel™. Significant hypoxia-selective downregulation of pseudo-vessels formation by Ru-Isq correlates with efficient inhibition of cell motility. Interestingly, in the applied concentration doses migration of endothelial cells was also inhibited by NAMI-A, but the pseudo-vessels formation on Matrigel™ was unaffected. Angiogenesis-related genes expression profile for both mature and precursor ECs indicated that inhibition of angiogenesis, mainly due to Ru-Isq, as compared to NAMI-A and Ru-Ind correlated with downregulation of CD31 and CD144 expression and upregulation of NOTCH4 expression in mature ECs, which is essential for endothelial cell motility and stalk cells organization control. The hypoxia-selective antiangiogenic activity of Ru-Ind and Ru-Isq, NAMI-A analogues makes them potent antimetastatic therapeutics for their selective action in hypoxia which controls tumor pathologic angiogenesis.

Lactoperoxidase (LPO) together with its (pseudo)halogenation cycle substrates, H₂O₂ and thiocyanate ions oxidized to hypothiocyanite ions, form one of the main systems involved in antimicrobial defense within the oral cavity. In bacterial diseases such as dental caries, lactoperoxidase is oxidized to a form known as Compound II, which is characterized by its inability to oxidize SCN^–, resulting in a decreased generation of antimicrobial products. Reynoutria sp. rizome extracts, due to their high polyphenol content, have been tested as a source of compounds able to regenerate the antimicrobial activity of lactoperoxidase through converting the Compound II to the native LPO state. In the presented study, acetone extracts of R. japonica, R. sachalinensis, and R. x bohemica, together with their five fractions and four selected polyphenols dominating in the studied in extracts, were tested toward lactoperoxidase reactivating potential. For this purpose, IC50, EC50, and activation percentage were determined by Ellman’s method. Furthermore, the rate constants for the conversion of Compound I–Compound II and Compound II–native-LPO in the presence of extracts, extracts fractions, and selected polyphenols were determined. Finally, the ability to enhance the antimicrobial properties of the lactoperoxidase system was tested against Streptococcus mutans. We proved that Reynoutria sp. rhizome is the source of lactoperoxidase peroxidation cycle substrates, which can act as activators and inhibitors of the antimicrobial properties of that system. The presented study shows that the reactivation of lactoperoxidase could become a potential therapeutic target in prevention and treatment support in some infectious oral diseases.