In the last decade, several studies from different fields have contributed significantly to the development of the enzyme biofuel cell (BFC). Moreover, the design of biocatalyst‐modified electrodes is a model for the development of synthetic and biomimetic electrocatalysts. The revival of the interest in the old BFC idea developed in the early 20th century is related to the development of new electrode materials as well as to the potential application of enzyme BFC miniaturisation in implantable bioelectronics. Furthermore, fundamental studies on half‐cells and electron transfer involving protein film electrodes performed in the last few decades have established new types of electrodes for BFC applications. In terms of applicability, some research groups have sought to obtain unit cells with different properties and characteristics such as membrane‐less BFCs by using flexible and low‐costing materials; in addition, micrometric BFCs, which can be implanted in different body parts, have also been developed to supply electrical power to bioelectronics devices. Thus, in this review, we focus on introducing the main concepts and challenges in enzyme BFC research. Some important topics in kinetics and thermodynamics applied to BFCs are also discussed. Wherever possible, an extensive review of works undertaken in recent years is performed. The topics are organised into three major themes: thermodynamics, kinetics and challenges in applicability.

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NAD-dependent alcohol dehydrogenase (ADH) enzymes for ethanol oxidation were investigated by differential electrochemical mass spectrometry (DEMS). The broad mass spectra obtained under bioelectrochemical control and with unprecedented accuracy were used to provide new insight into the enzyme kinetics and mechanisms.

Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans) where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

Endonuclease III (EndoIII) is a DNA glycosylase that contains the [4Fe4S] cluster, which is essential for the protein to bind to damaged DNA in a process called base excision repair (BER). Here we propose that the change in the covalency of Fe-S bonds of the [4Fe4S] cluster caused by double-stranded (ds)-DNA binding is accompanied by a change in their strength, which is due to alterations of the electronic structure of the cluster. Micro-FTIR spectroscopy in the mid-IR region and FTIR spectroscopy in the far IR (450 and 300 cm −1) were used independently to study the structural changes in EndoIII and the behavior of the [4Fe4S] cluster it contains, in the native form and upon its binding to ds-DNA. Structural changes in the DNA itself were also examined. The characteristics vibrational modes, corresponding to Fe-S (sulfide) and Fe-S (thiolate) bonds were identified in the cluster through far IR spectroscopy as well through quantum chemistry calculations. Based on the experimental results, these vibrational modes shift in their spectral positions caused by negatively charged DNA in the vicinity of the cluster. Modifications of the Fe-S bond lengths upon DNA binding, both of the Fe-S (sulfide) and Fe-S (thiolate) bonds in the [4Fe4S] cluster of EndoIII are responsible for the stabilization of the cluster towards higher oxidation state (3+), and hence its redox communication along the ds-DNA helix. Metalloproteins containing Fe-S clusters constitute an important group due to several important functions including electron transfer, H 2 evolution, regulation, and catalysis 1,2. In particular, there are proteins actively involved in the base excision repair (BER) pathway, interacting with DNA for the search of oxidized/damaged bases and its consequently excising them from the genome 3-5. The E. coli DNA glycosylases EndoIII and MutY are homologues and are involved in the removal of damaged pyrimidines and the removal of mismatched adenine from the A:8-oxoguanine (8-oxoG) pair, respectively 6,7. Among the six different superfamilies of DNA glycosylases, EndoIII, a bifunctional DNA glycosylase, was the first-discovered enzyme. It belongs to one of the two superfamilies, called helix-hairpin-helix (HhH), which contain Fe-S clusters 7,8. This name was assigned on the basis of the secondary structural element present in these enzymes that is essential for DNA binding 9. Other members of this superfamily include MutY, Endonuclease III-like protein 1 (hNTHL1), human MutY homolog DNA glycosylase (MUTYH), and 3-methyladenine glycosylase II (AlkA) 10,11. Earlier characterization revealed that both EndoIII and MutY contain [4Fe4S] clusters 12 ; initially, however, it was not easy to show that the cluster is redox active under physiological conditions, and hence it was basically assigned a structural role 13. But the quest to prove that these DNA glycosylases detect a single damaged base within an enormous number of normal bases and the specific role of Fe-S clusters in these processes has led to several important revelations abou...

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