Fast electron transfer through a single molecule natively structured redox protein

Abstract

The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conductance through single-molecules of the electron transfer protein cytochrome b 562 in its native conformation, under pseudo-physiological conditions. This is achieved by thiol (SH) linker pairs at opposite ends of the molecule through protein engineering, resulting in defined covalent contact between a gold surface and a platinum-iridium STM tip. Two different orientations of the linkers were examined: a long-axis configuration (SH-LA) and a short-axis configuration (SH-SA). In each case, the molecular conductance could be 'gated' through electrochemical control of the heme redox state. Reproducible and remarkably high conductance was observed in this relatively complex electron transfer system, with single-molecule conductance values peaking around 18 nS and 12 nS for the SH-SA and SH-LA cytochrome b 562 molecules near zero electrochemical overpotential. This strongly points to the important role of the heme co-factor bound to the natively structured protein. We suggest that the two-step model of protein electron transfer in the STM geometry requires a multi-electron transfer to explain such a high conductance. The model also yields a low value for the reorganisation energy, implying that solvent reorganisation is largely absent.

Redox tuning of cytochrome b562 through facile metal porphyrin substitution

“…7 Using engineered proteins in conjunction with single molecule STM studies has allowed us to demonstrate that cyt b 562 is highly conductive with conductance being electrochemically gated. 10 The ability to tune the redox potentials and add different conducting centres would thus expand the application of cyt b 562 and improve our fundamental understanding of metal-protein interactions. There has been some success in varying the redox potential of cyt b 562 through conventional protein mutagenesis by tuning the protein's interactions with the heme group.…”

Switching metalloporphyrin binding specificity of a b-type cytochrome to fluorogenic zinc by design

“…Cyt b562 ( Figure S1) is a small, helical bundle protein that binds haem non-covalently [22][23][24] . It is an important model for electron transfer [25][26][27][28] , engineering novel components [29][30][31][32][33][34] and assembling supramolecular structures 6,14,35 . ZnPP can replace haem through passive exchange 20,33,36 but the environment is non-ideal resulting in relatively low affinity binding (404 nM ±11 KD; Table S1) and a significant background haem binding in vivo (Figure 1).…”

Voltammetry and molecular assembly of G-quadruplex DNAzyme on single-crystal Au(111)-electrode surfaces – hemin as an electrochemical intercalator

“…Electronic conductivity of redox metalloproteins and metalloenzymes 34,[36][37][38][39]55,56 , and of double strand DNA-based molecules [11][12][13][16][17][18][19]57,58 immobilized at electrochemical interfaces have been explored comprehensively. Electronic conductivity of "non-canonical" DNA forms, quadruplexes in particular have also been addressed, but such reports have only begun to appear much more recently [27][28][29][30][31][32][33] , and with little if any direct approach to interfacial ; (c) surface immobilized G-quadruplexes finally offer perspectives for ultra-sensitive detection of metal ions [40][41][42] , complex biomolecules such as peptides [23][24][25][26][27][28][29][30] , and perhaps other analytes.…”

“…These studies include the use of a redox probe intercalated or covalently bound to the OGN frame and electron exchange between the redox probe and the electrode. [36][37][38][39] . Other recent studies have addressed single-molecule conductivity in situ using direct conductivity approaches 39 .…”