On the Structure and Reaction Mechanism of Human Acireductone Dioxygenase

• Published in: Chemistry : a European journal 2018-04, Vol.24 (20), p.5225-5237
• Main Authors: Miłaczewska, Anna, Kot, Ewa, Amaya, José A., Makris, Thomas M., Zając, Marcin, Korecki, Józef, Chumakov, Aleksandr, Trzewik, Bartosz, Kędracka‐Krok, Sylwia, Minor, Władek, Chruszcz, Maksymilian, Borowski, Tomasz
• Format: Article
• Language: English
• Subjects: acireductone dioxygenase; Binding; Catalysis; Catalytic Domain; Chemistry; Computation; Computer applications; Crystal structure; Dioxygenase; Dioxygenases - chemistry; Enzymes; EPR spectroscopy; Humans; Ions; Iron; Iron - chemistry; Metal ions; Metals; Methionine; Models, Molecular; Mössbauer spectroscopy; Oxidation; Oxidation-Reduction; Protein Binding; Protein Conformation; protein structures; Quercetin; Reaction mechanisms; Salvage; Selenomethionine; Selenomethionine - chemistry; Signal Transduction; Spectroscopy; Spectrum analysis; Substrates; Vibrational spectra
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201704617

Acireductone dioxygenase (ARD) is an intriguing enzyme from the methionine salvage pathway that is capable of catalysing two different oxidation reactions with the same substrate depending on the type of the metal ion in the active site. To date, the structural information regarding the ARD–acireductone complex is limited and possible reaction mechanisms are still under debate. The results of joint experimental and computational studies undertaken to advance knowledge about ARD are reported. The crystal structure of an ARD from Homo sapiens was determined with selenomethionine. EPR spectroscopy suggested that binding acireductone triggers one protein residue to dissociate from Fe2+, which allows NO (and presumably O2) to bind directly to the metal. Mössbauer spectroscopic data (interpreted with the aid of DFT calculations) was consistent with bidentate binding of acireductone to Fe2+ and concomitant dissociation of His88 from the metal. Major features of Fe vibrational spectra obtained for the native enzyme and upon addition of acireductone were reproduced by QM/MM calculations for the proposed models. A computational (QM/MM) study of the reaction mechanisms suggests that Fe2+ promotes O−O bond homolysis, which elicits cleavage of the C1−C2 bond of the substrate. Higher M3+/M2+ redox potentials of other divalent metals do not support this pathway, and instead the reaction proceeds similarly to the key reaction step in the quercetin 2,3‐dioxygenase mechanism. hARD working enzyme! Experimental and computational studies of acireductone dioxygenase (ARD) from the methionine salvage pathway suggested reaction mechanisms catalysed by two isoforms: Fe2+ promotes O−O homolysis and subsequent C1−C2 bond cleavage of the acireductone substrate, whereas Ni2+ promotes a concerted O−O, C1−C2 and C2−C3 bond heterolysis (see figure).