Frattini Gianfranco, Puzzolo Juan L, Drusin Salvador I, Mansilla Santiago, Radi Rafael, Demicheli Verónica, Moreno Diego M
Instituto de Química Rosario (CONICET-UNR) and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina.
Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Uruguay.
Arch Biochem Biophys. 2025 Jul;769:110439. doi: 10.1016/j.abb.2025.110439. Epub 2025 Apr 25.
Manganese superoxide dismutase (MnSOD) is a critical enzyme responsible for detoxifying superoxide radicals in mitochondria, thereby ensuring oxidative balance within cells. Post-translational modifications (PTMs), such as acetylation and nitration, significantly influence MnSOD's catalytic efficiency. Site-specific nitration of MnSOD at tyrosine 34 by peroxynitrite leads to an irreversible inactivation, which has been widely observed in diverse pathologies. On the other hand, acetylation of MnSOD is a reversible modification that modulates the activity of the enzyme and it is finely regulated by the action of the protein Sirt3, responsible for the deacetylation of a wide variety of mitochondrial enzymes. This study focuses on Lys29 acetylation and its impact on the enzyme's activity and its interplay with peroxynitrite-mediated nitration of Tyr34. Through molecular dynamics (MD) simulations, we observed that acetylation of Lys29 partially obstructs the access channel to the active site, reducing superoxide accessibility. Electrostatic potential calculations further revealed that Lys29 acetylation diminishes the positive charge around the active site, contributing to decreased affinity for superoxide radicals. Brownian dynamics (BD) simulations confirmed a 50 % reduction in the enzyme's association rate constant (k) for superoxide upon Lys29 acetylation. In contrast, Lys98 acetylation had a minor effect on k. In vitro studies also supported our findings and showed that acetylation could play a role in the irreversible inactivation of MnSOD by peroxynitrite, likely by sterically hindering Tyr34 nitration. These findings highlight the role of acetylation as a reversible protective mechanism that can regulate superoxide and peroxynitrite accessibility to MnSOD under stress conditions.
锰超氧化物歧化酶（MnSOD）是一种关键酶，负责清除线粒体中的超氧自由基，从而确保细胞内的氧化平衡。翻译后修饰（PTM），如乙酰化和硝化，会显著影响MnSOD的催化效率。过氧亚硝酸盐对MnSOD酪氨酸34位点的特异性硝化会导致不可逆失活，这在多种病理情况下已被广泛观察到。另一方面，MnSOD的乙酰化是一种可逆修饰，可调节该酶的活性，并且受到蛋白质Sirt3作用的精细调控，Sirt3负责多种线粒体酶的去乙酰化。本研究聚焦于赖氨酸29的乙酰化及其对酶活性的影响，以及它与酪氨酸34的过氧亚硝酸盐介导的硝化之间的相互作用。通过分子动力学（MD）模拟，我们观察到赖氨酸29的乙酰化部分阻碍了活性位点的通道，降低了超氧的可及性。静电势计算进一步表明，赖氨酸29的乙酰化减少了活性位点周围的正电荷，导致对超氧自由基的亲和力降低。布朗动力学（BD）模拟证实，赖氨酸29乙酰化后，该酶与超氧的结合速率常数（k）降低了50%。相比之下，赖氨酸98的乙酰化对k的影响较小。体外研究也支持了我们的发现，并表明乙酰化可能在过氧亚硝酸盐导致的MnSOD不可逆失活中起作用，可能是通过空间位阻阻碍酪氨酸34的硝化。这些发现突出了乙酰化作为一种可逆保护机制的作用，在应激条件下可调节超氧和过氧亚硝酸盐对MnSOD的可及性。
