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An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (RRMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.

The basidiomycete Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper), which is a new type of pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH; EC 1.1.99.18) from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km) of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall.

Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes.