Electrocatalysis by Heme Enzymes—Applications in Biosensing

Abstract

Heme proteins take part in a number of fundamental biological processes, including oxygen transport and storage, electron transfer, catalysis and signal transduction. The redox chemistry of the heme iron and the biochemical diversity of heme proteins have led to the development of a plethora of biotechnological applications. This work focuses on biosensing devices based on heme proteins, in which they are electronically coupled to an electrode and their activity is determined through the measurement of catalytic currents in the presence of substrate, i.e., the target analyte of the biosensor. After an overview of the main concepts of amperometric biosensors, we address transduction schemes, protein immobilization strategies, and the performance of devices that explore reactions of heme biocatalysts, including peroxidase, cytochrome P450, catalase, nitrite reductase, cytochrome c oxidase, cytochrome c and derived microperoxidases, hemoglobin, and myoglobin. We further discuss how structural information about immobilized heme proteins can lead to rational design of biosensing devices, ensuring insights into their efficiency and long-term stability.

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices

“…However, the progress in the development of peroxidase-based applications is lagging behind the expectations, due to the difficulties in transferring the enzyme catalytic efficiency in solution to the immobilised state and the lack of experimental approaches for fast screening of enzyme/electrode constructs in situ [ 30 ]. The enzyme structural integrity, its redox properties and reversibility of the redox reaction, catalytic efficiency and stability need to be preserved upon immobilisation, and the efficient electronic communication with the electrode has to be ensured [ 7 , 30 ]. Therefore, sensitive methods that can probe these features to allow for a rational design of peroxidase-based devices with optimised performance, are highly required [ 21 , 29 ].…”

“…Sci. 2021, 22, 7998 2 of 15 and catalytic parameters of the biocatalyst upon immobilisation, efficient DET between the enzyme and the electrode, good substrate accessibility to the active site of the enzyme and a long-term stability of the enzyme/electrode construct [7]. The only experimental approach that allows for the simultaneous and thorough structural characterization and redox potential determination of the immobilised enzyme in situ relies on surface-enhanced resonance Raman (SERR) spectroelectrochemistry.…”

“…These structural changes that frequently occur upon attachment of the enzyme to electrodes are actually the most common cause for the failure of 3 rd -generation devices [14]. The catalytic performance of immobilised enzymes can be simultaneously monitored by electrochemical methods [7]. This experimental approach that couples SERR with electrochemistry has been used to evaluate the potential of several interesting enzymes for the development of biotechnological applications; these include cytochrome P450, nitrite reductases, microperoxidases and, more recently, DyP peroxidases [13][14][15][16][17][18][19][20].…”

“…They rely on enzymes immobilised on biocompatible electrodes that undergo direct electron transfer (DET) with the electrode (i.e., electrochemical transducer), which acts as a source or a sink of electrons that drives the catalytic reaction in the presence of the substrate [ 5 , 6 ]. Crucial parameters that govern a rational design of 3 rd -generation bioelectronic devices are the preservation of the solution structure, redox potential (E 0 ) and catalytic parameters of the biocatalyst upon immobilisation, efficient DET between the enzyme and the electrode, good substrate accessibility to the active site of the enzyme and a long-term stability of the enzyme/electrode construct [ 7 ]. The only experimental approach that allows for the simultaneous and thorough structural characterization and redox potential determination of the immobilised enzyme in situ relies on surface-enhanced resonance Raman (SERR) spectroelectrochemistry.…”

Flat, Ferrocenyl‐Conjugated Peptides: A Combined Electrochemical and Spectroscopic Study

“…Electron/charge transfer in biological molecules is a crucial event for life [1] . In addition, by unraveling these natural mechanisms one can design systems with new functions, such as enzymatic fuel cells, [2] biosensors, [3] or bio‐electrocatalysts [4] . To this aim, the study of simpler, synthetic peptides provides an easy‐to‐handle tool to access the mechanisms of electron transfer in proteins.…”