Abstract：
:PQQ is an exogenous, tricyclic, quino-cofactor for a number of bacterial dehydrogenases. The final step of PQQ formation is catalyzed by PqqC, a cofactorless oxidase. This study focuses on the activation of molecular oxygen in an enzyme active site without metal or cofactor and has identified a specific oxygen binding and activating pocket in PqqC. The active site variants H154N, Y175F,S, and R179S were studied with the goal of defining the site of O(2) binding and activation. Using apo-glucose dehydrogenase to assay for PQQ production, none of the mutants in this "O(2) core" are capable of PQQ/PQQH(2) formation. Spectrophotometric assays give insight into the incomplete reactions being catalyzed by these mutants. Active site variants Y175F, H154N, and R179S form a quinoid intermediate (Figure 1) anaerobically. Y175S is capable of proceeding further from quinoid to quinol, whereas Y175F, H154N, and R179S require O(2) to produce the quinol species. None of the mutations precludes substrate/product binding or oxygen binding. Assays for the oxidation of PQQH(2) to PQQ show that these O(2) core mutants are incapable of catalyzing a rate increase over the reaction in buffer, whereas H154N can catalyze the oxidation of PQQH(2) to PQQ in the presence of H(2)O(2) as an electron acceptor. Taken together, these data indicate that none of the targeted mutants can react fully to form quinone even in the presence of bound O(2). The data indicate a successful separation of oxidative chemistry from O(2) binding. The residues H154, Y175, and R179 are proposed to form a core O(2) binding structure that is essential for efficient O(2) activation.
journal_name
Biochemistryjournal_title
Biochemistryauthors
RoseFigura JM,Puehringer S,Schwarzenbacher R,Toyama H,Klinman JPdoi
10.1021/bi1015474subject
Has Abstractpub_date
2011-03-08 00:00:00pages
1556-66issue
9eissn
0006-2960issn
1520-4995journal_volume
50pub_type
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