No abstract

Superoxide reductase (SOR) is a non-heme iron metalloenzyme that detoxifies superoxide radical in microorganisms. Its active site consists of an unusual non-heme Fe(2+) center in a [His4Cys1] square pyramidal pentacoordination, with the axial cysteine ligand proposed to be an essential feature in catalysis. Two NH peptide groups from isoleucine 118 and histidine 119 establish hydrogen bonds involving the sulfur ligand (Desulfoarculus baarsii SOR numbering). To investigate the catalytic role of these hydrogen bonds, the isoleucine 118 residue of the SOR from Desulfoarculus baarsii was mutated into alanine, aspartate, or serine residues. Resonance Raman spectroscopy showed that the mutations specifically induced an increase of the strength of the Fe(3+)-S(Cys) and S-Cβ(Cys) bonds as well as a change in conformation of the cysteinyl side chain, which was associated with the alteration of the NH hydrogen bonding involving the sulfur ligand. The effects of the isoleucine mutations on the reactivity of SOR with O2 (•-) were investigated by pulse radiolysis. These studies showed that the mutations induced a specific increase of the pK a of the first reaction intermediate, recently proposed to be an Fe(2+)-O2 (•-) species. These data were supported by density functional theory calculations conducted on three models of the Fe(2+)-O2 (•-) intermediate, with one, two, or no hydrogen bonds involving the sulfur ligand. Our results demonstrated that the hydrogen bonds between the NH (peptide) and the cysteine ligand tightly control the rate of protonation of the Fe(2+)-O2 (•-) reaction intermediate to form an Fe(3+)-OOH species.

3 pagesInternational audienceHere we uncover a key amino acid at the entrance of the active site of glucose oxidase from Penicilium amagasakiense and we successfully redesign it by nonactive site mutations, leading to enzymatic anodes with 2.4 fold higher current densities. The increase in current density could be correlated with a better interaction between the negatively charged amino acid located in position 424 and the positively charged redox mediator. We also demonstrate that this increase in current was not specific to Osmium and that further introduction of negative charges around the active site of glucose oxidase lead to a decrease in current density, illustrating the uniqueness of this amino acid in position 42

Superoxide reductase (SOR), a non-heme mononuclear iron protein that is involved in superoxide detoxification in microorganisms, can be used as an unprecedented model to study the mechanisms of O2 activation and of the formation of high-valent iron-oxo species in metalloenzymes. By using resonance Raman spectroscopy, it was shown that the mutation of two residues in the second coordination sphere of the SOR iron active site, K48 and I118, led to the formation of a high-valent iron-oxo species when the mutant proteins were reacted with H2O2. These data demonstrate that these residues in the second coordination sphere tightly control the evolution and the cleavage of the O-O bond of the ferric iron hydroperoxide intermediate that is formed in the SOR active site.

In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.

The O2 reactivity of flavoproteins and particularly, glucose oxidase, is a limitation for numerous biotechnological applications, and this has been the subject of intense research. In the present study, we performed site‐directed mutations on Val464 of GOx from Penicilium amagasakiense and combined steady‐state, rapid kinetics and electrochemistry to investigate the effect of such mutations on the O2 reactivity. We propose that replacing the non‐polar Valine with a polar Serine permits to decrease the diffusion/stabilization of O2 near the active redox center without altering the oxidation of glucose. When incorporated in osmium hydrogels, the resulting electrodes were insensitive to O2 in the 0.5–60 mM glucose concentration range and were 4.7 times more stable under continuous operation than similar electrodes made with the native GOx. To the best of our knowledge, this is the first example in which O2 sensitivity in GOx can be significantly decrease without altering the electrochemical efficiency for glucose oxidation.

superoxide radical ͉ hydrogen peroxide

Superoxide reductase (SOR), a non-heme mononuclear iron protein that is involved in superoxide detoxification in microorganisms, can be used as an unprecedented model to study the mechanisms of O 2 activation and of the formation of high-valent iron-oxo species in metalloenzymes. By using resonance Raman spectroscopy, it was shown that the mutation of two residues in the second coordination sphere of the SOR iron active site, K 48 and I 118 , led to the formation of a high-valent iron-oxo species when the mutant proteins were reacted with H 2 O 2 . These data demonstrate that these residues in the second coordination sphere tightly control the evolution and the cleavage of the O À O bond of the ferric iron hydroperoxide intermediate that is formed in the SOR active site.