Electrical contacting of an assembly of pseudoazurin and nitrite reductase using DNA-directed immobilization.
A method for the electrical contacting of redox enzymes that obtain oxidizing or reducing equivalents from small electron-transfer proteins is demonstrated. The electrochemical contacting of redox enzymes through their immobilization onto electrode supports offers great potential for technological applications and for fundamental studies, but finding appropriate methods to immobilize the enzymes in an orientation allowing rapid electron transfer with the electrode has proven difficult. The copper enzyme nitrite reductase (NiR) and its natural electron-exchange partner pseudoazurin (Paz) are conjugated to a specific DNA tag and immobilized to a gold electrode into a stoichiometrically defined assembly. The DNA tethered to the electrode surface acts as flexible place-holder for the protein components, allowing both proteins to move within the construct. It is shown that Paz efficiently shuttles electrons between the electrode and the NiR enzyme, allowing the electrochemically driven NiR catalysis to be monitored. The activity of the NiR enzyme remains unperturbed by the immobilization. The rate-limiting step of the system is tentatively ascribed to the dissociation of the Paz/NiR complex. The electrochemical response of the system reports not only on the NiR catalysis and on interfacial electron transfer but also on the interaction between NiR and Paz.
Tepper
Armand W J W
AW
Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands. w.tepper@chem.leidenuniv.nl
D000421:Alcaligenes / Q000201:enzymology; D001400:Azurin / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D055162:Biocatalysis; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D004566:Electrodes; D004579:Electron Transport; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009994:Osmolar Concentration; D011487:Protein Conformation; D055672:Static Electricity; D013499:Surface Properties
D004800:Enzymes, Immobilized; D009573:Nitrites; C047163:pseudoazurin; D001400:Azurin; D009569:Nitric Oxide; D004247:DNA; D009572:Nitrite Reductases