In a new study, researchers from Japan have developed a novel hybrid protein complex by binding lysozyme to copper, which has enhanced superoxide dismutase activity that can remove ROS more effectively. These complexes could find use in therapeutic drug applications.
Newswise — In aerobic living beings, reactive oxygen species (ROS), like hydroxide (OH), singlet oxygen (1O2), hydrogen peroxide (H2O2), and superoxide (O2–) ions emerge throughout aerobic respiration, leading to severe oxidative harm to biomolecules within the organism. Thus, eliminating ROS, particularly O2–, holds utmost significance as it combines with H+ to generate additional hazardous ROS species such as H2O2 and OH.
This is accomplished by metalloenzymes referred to as superoxide dismutases (SODs). These enzymes contain metallic ions (such as Ni, Fe, Mn, Cu, and Zn) within their active sites, which facilitate the breakdown of O2– into H2O2 and O2. In this context, small-sized Cu(II) complexes have become significant as efficient SOD analogues that demonstrate notable SOD capability. Nevertheless, they are constrained by their inclination to become harmful to biomolecules following the liberation of Cu(II).
In a recent investigation, an assemblage of scientists led by Assistant Professor Daisuke Nakane and Professor Takashiro Akitsu from the Chemistry Department at Tokyo University of Science (TUS) has created a groundbreaking metal-protein hybrid compound with augmented ROS functionality. They merged the hydrolytic enzyme lysozyme with a Cu(II) complex possessing SOD-like properties, resulting in the hybrid formation known as lysozyme CuST@lysozyme. This hybrid exhibited encouraging SOD activity while maintaining low levels of biotoxicity.
"The focus of our study was to examine the synthesis of a hybrid protein combining lysozyme and a Cu(II) complex that mimics the functionality of SOD. We selected lysozyme for its inherent stability and crystalline nature. Our hypothesis was that this hybrid protein with SOD-mimetic properties would enhance the biocompatibility and stability of the Cu(II) complex acting as a functional SOD model," explains Dr. Nakane, providing the rationale for their research. The study, published on 27 April 2023, can be found in Scientific Reports.
By employing comprehensive crystallographic and spectroscopic analysis, the research team, which included Assistant Professor Kenichi Kitanishi from TUS, Dr. Arshak Tsaturyan from Southern Federal University, Professor Masaki Unno from Ibaraki University, and other collaborators, successfully confirmed the formation of the hybrid protein CuST@lysozyme and unraveled its structure. Their findings revealed that the His15 imidazole group of lysozyme binds to the Cu(II) center of CuST in the equatorial position, while the CuST unit is stabilized axially by multiple weak coordination and hydrogen bonds. Additionally, they suggested the potential coordination of O2– to the Cu(II) center. Through various assays, the researchers demonstrated the remarkable SOD activity and stability of the biocompatible CuST@lysozyme hybrid protein complex.
Based on their thorough spectroscopic analysis and quantum calculations, the team has put forth a proposed five-step mechanism for the O2– disproportionation process mediated by the complex. These steps include (1) the Cu(II) resting state, (2) the Cu(II) state with bound O2–, (3) the Cu(I) resting state achieved through protonation of the carboxylate ligand, (4) the Cu(I) state interacting with O2–, and (5) the Cu(II) state interacting with H2O2. Furthermore, they suggest potential strategies for enhancing the stability of the complex. These include inhibiting ligand dissociation through the utilization of late-transition-metal complexes for binding to lysozyme, increasing the interaction between the complexes and lysozyme through the incorporation of ligands with hydrogen-bonding functionalities, and introducing acidic functional groups to counterbalance the basic side chains of lysozyme.
The study presents a novel category of biocompatible hybrid protein complexes with SOD activity, which exhibit no adverse reactions with bodily fluids following the decomposition of the mimetic complex. "We have strategically enhanced the stability of the metal-lysozyme composites, particularly in biological fluids like plasma and cytosol. This advancement is expected to stimulate further discussions regarding their potential therapeutic applications," concludes Prof. Akitsu.
We will certainly benefit from advancements like these that add to our repertoire of biocompatible complexes for advanced therapeutics.
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