On the Structure and Reaction Mechanism of Human Acireductone Dioxygenase

Abstract

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 Fe , which allows NO (and presumably O ) to bind directly to the metal. Mössbauer spectroscopic data (interpreted with the aid of DFT calculations) was consistent with bidentate binding of acireductone to Fe 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 Fe promotes O-O bond homolysis, which elicits cleavage of the C1-C2 bond of the substrate. Higher M /M 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.

Second‐Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

“…Accordingly, in intradiol and extradiol catechol dioxygenases the substrate binds as a bidentate ligand in the equatorial plane with dioxygen in the distal position, whereas the remaining ligands of iron are side chains of a glutamic acid and three histidine groups although sometimes a tyrosinate ligand is also included . In a similar vein, in the nonheme iron enzyme acireductone dioxygenase the substrate binds the iron as a bidentate ligand in the equatorial plane and reacts with dioxygen directly on the iron center …”

The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase

“…The complete details of these structures have not been published and it is unknown if the proteins used in these structures were enzymatically active, nor have the identities of the metals in the active sites been confirmed. 61–62 In the B. anthracis ARD (BaARD) structure, the bound metal is proposed to be Cd 2+ , while in the HsARD structure, the bound metal ion is proposed to be Fe 3+ . This is somewhat surprising, in that in our hands, oxidation results in the loss of metal and inactivation of the enzyme.…”

“…The recently deposited structure of Fe 3+ -HsARD (4QGN) has L-selenomethionine in the active site and it is coordinated to the metal ion in a manner similar to that of D-LA. 62 …”

Structure, function, and biosynthesis of nickel‐dependent enzymes

“…This activity has been confirmed in vitro for mammalian Mus musculus and human ARD. However, it is unlikely that NiARD plays a biologically relevant role in eukaryotes . While the Fe‐containing form uses Fe(II) to activate dioxygen for the oxidation of the substrate via a redox chemistry (oxidative cleavage of C1C2 bond), Ni(II) is used as a Lewis acid to activate the substrate toward reaction with O 2 (cleavage of C1C2 and C2C3 bonds).…”