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| Author(s): |
Arthur Migliatti Vetorazzi Ferreira de Pinho
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2025-01-30 |
| Examining board members: |
Daniela Ramos Truzzi;
Marcos Antonio de Oliveira;
José Carlos Toledo Junior
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| Advisor: | Daniela Ramos Truzzi |
| Abstract | |
Nitric oxide (NO) is a radical molecule produced endogenously and has various biological functions, including vasodilation, neurotransmission, and defense against invading organisms. In biological environments, the most abundant metabolite of NO is the iron dinitrosyl complex (DNIC). In addition to the two NO fragments, DNICs also contain two other ligands, which in cells are predominantly thiol-containing molecules of high molecular weight, such as proteins. Although the identity of these proteins has not yet been fully clarified, preliminary studies conducted in our laboratory suggest that those that bind through two amino acid residues form more stable DNICs. Studies in the literature indicate that increased NO concentrations in human mammary epithelial tumor cells lead to a decrease in H2O2 metabolism, and it is known that under these conditions, DNIC formation is also favored. With this in mind, thioredoxin 1 (Trx1) emerges as a relevant protein, as its active site consists of two cysteine residues separated by only two amino acid residues, and it is involved in hydrogen peroxide (H2O2) metabolism. Therefore, the objective of this work was to investigate the ability of Saccharomyces cerevisiae Trx1 (yTrx1) to form DNIC (DNIC-yTrx1) in aqueous solution, characterize the formed complex, and assess the impact of this formation on the reductase activity of yTrx1. To achieve this, we expressed and purified yTrx1 and synthesized DNIC-GS using two synthesis methods. We demonstrated through high-performance liquid chromatography that one of the methods resulted in a formation of oxidant molecules at more than twice the concentration, which could impact the yield of DNIC-yTrx1 formation. We then analyzed the reaction kinetics of yTrx1 with DNIC-GS using Electron Paramagnetic Resonance (EPR), obtaining an axial signal (g⊥ = 2.038 and g|| = 2.022) and estimated the second-order rate constant (k ~ 70 M-1s-1). We also estimated the dissociation constant (Kd ~ (1.4 ± 0.9) × 10-1 M-1) for the DNIC-yTrx1 complex. In the analysis of DNIC-yTrx1 by electronic absorption spectroscopy, we observed the formation of two peaks at wavelengths 335 nm (ε = 4308 ± 149 M-1 cm-1) and 400 nm (ε = 3250 ± 140 M-1 cm-1). Fluorescence spectroscopy revealed that DNIC-yTrx1 formation led to a 40% decrease in emission at 305 nm compared to free yTrx1. We performed native gel electrophoresis of the DNIC-yTrx1 solution, from which we confirmed the formation of a characteristic band for the complex. Initial EPR results indicated that DNIC-yTrx1 remained stable for 60 hours after the addition of BSA as a competing protein for yTrx1 binding to DNIC. Finally, we confirmed that the protein loses its reductase activity after the formation of DNIC-yTrx1. According to the data obtained in this work, yTrx1 is capable of binding to DNIC, forming a stable complex that results in the loss of its enzymatic activity. These findings may contribute to elucidating the experimental observations of decreased H2O2 metabolism as a result of increased NO via DNIC formation. (AU) | |
| FAPESP's process: | 22/11354-7 - Study of the interaction of dinitrosyl iron complexes (DNIC) and thioredoxins |
| Grantee: | Arthur Migliatti Vetorazzi Ferreira de Pinho |
| Support Opportunities: | Scholarships in Brazil - Master |