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.

Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a₃ in caa₃-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN⁻ ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells.

A dye-decolorizing peroxidase (DyP)-based electrochemical biosensor for hydrogen peroxide (H₂O₂) is developed in miniaturized, disposable, and user-friendly configuration. Wild type and variant DyPs are immobilized on self-assembled monolayer (SAM)-coated and nanostructure-modified screen-printed electrodes (SPEs) to ensure biocompatibility and increase the enzyme loading and hence the biosensor sensitivity. The structure of the enzymes attached to gold and silver nanoparticle (AuNP and AgNP)-modified carbon- and gold-based SPEs (C-SPE and Au-SPE) is monitored by resonance Raman spectroscopy and their electrocatalytic performance toward H₂O₂ by electrochemistry. Among the tested configurations, the wild type DyP/SAM/AuNP/C-SPE construct shows a superior performance, with a linear response range to H₂O₂ from 30 to 475 μM, a sensitivity of 234 ± 9 mA·M^-1·cm^-2, and a limit of detection (LOD) of 3.5 μM, measured in open air conditions. The device is suitable for single-use, on-site measurements of H₂O₂ in air-exposed samples such as physiological fluids. The herein developed biosensor shows a high potential for customization for the detection of other small hydroperoxide substrates and additional improvement of selected traits (e.g., sensitivity, thermal stability, and substrate inhibition properties) due to the easy and well-established production and genetic manipulation of the employed biocatalyst, DyP.

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H₂O₂. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H₂O₂. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates.