title: Orientational control over nitrite reductase on modified gold electrode and its effects on the interfacial electron transfer.
abstract: Recently, studies have been reported in which fluorescently labeled redox proteins have been studied with a combination of spectroscopy and electrochemistry. In order to understand the effect of the dye on the protein-electrode interaction, voltammetry and surface analysis have been performed on protein films of dye-labeled and unlabeled forms of a cysteine-surface variant (L93C) and the wild type (wt) of the copper containing nitrite reductase (NiR) from  Alcaligenes faecalis  S6. The protein has been adsorbed onto gold electrodes modified with self-assembled monolayers (SAMs) made up of 6-mercaptohexanol (6-OH) and mixtures of various octanethiols. Electrochemical and surface-analytical techniques were utilized to explore the influence of the SAM composition on wt and L93C NiR enzyme activity and the orientation of the enzyme molecules with respect to the electrode/SAM. The unlabeled L93C NiR enzyme is only electroactive on mixed SAMs composed of positive 8-aminooctanethiol (8-NH(2)) and 8-mercaptooctanol (8-OH). No enzymatic activity is observed on SAMs consisting of pure 6-OH, 8-OH, or pure 8-NH(2). Modification of L93C NiR with the ATTO 565 dye resulted in enzymatic activity on SAMs of 6-OH, but not on SAMs of 8-OH. Quartz crystal microbalance with dissipation measurements show that well-ordered and rigid protein films (single orientation of the protein) are formed when NiR is electroactive. By contrast, electrode-NiR combinations for which no electrochemical activity is observed still have NiR adsorbed on the surfaces, but a less-structured and water-rich film is formed. For the unlabeled L93C NiR, bilayer formation is observed, suggesting that the Cys93 residue is orientated away from the surface and able to form disulfide bridges to a second layer of L93C NiR. The results indicate that interfacial electron transfer is only possible if the negatively charged surface patch surrounding the electron-entry site of NiR is directed toward the electrode. This can be achieved either by introducing positive charges in the SAM or, when the SAM does not carry a charge, by labeling the enzyme with an ATTO 565 dye, which has some hydrophobic character, close to the electron entry site of the NiR.
authors.affiliation: Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Krzemiński
authors.forename: Samuel
authors.initials: S
authors.lastname: Cronin
authors.forename: Lionel
authors.initials: L
authors.lastname: Ndamba
authors.forename: Gerard W
authors.initials: GW
authors.lastname: Canters
authors.forename: Thijs J
authors.initials: TJ
authors.lastname: Aartsma
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Evans
authors.forename: Lars J C
authors.initials: LJ
authors.lastname: Jeuken
mesh_terms: D000421:Alcaligenes / Q000201:enzymology; D055664:Electrochemical Techniques; D004566:Electrodes; D004579:Electron Transport; D005456:Fluorescent Dyes / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D058525:Quartz Crystal Microbalance Techniques; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D006046:Gold; D009572:Nitrite Reductases