Bioelectrochemical Characterization of a Molybdenum-Containing Aldehyde Dehydrogenase Variant Deleting Its Cytochrome C Subunit
Authors: Ichikawa Konatsu , Adachi Taiki , Sowa Keisei , Kitazumi Yuki , Shirai Osamu 
Published: December 2024

Bioelectrocatalysis is a coupled system with enzymatic and electrode reactions. Some oxidoreductases can directly communicate with electrodes, which is called direct electron transfer (direct ET; DET)-type bioelectrocatalysis. The number of enzymes proceeding with DET-type reactions is still limited, and its mechanism is not fully elucidated. However, it is expected to be applied to electrochemical devices such as biosensors, biofuel cells, and bioreactors, owing to energy efficiency, biocompatibility, and design flexibility. In addition, this reaction is utilized for enzyme characterization based on kinetics and thermodynamics. In particular, a relationship between DET activity and the redox potential can be quickly and precisely characterized.
We focused on aldehyde dehydrogenase (ALDH) from Gluconobacter oxydans, a membrane-bound protein catalyzing DET-type acetaldehyde oxidation. In previous studies, the three-dimensional structure of ALDH was elucidated using cryo-electron microscopy analysis (PDB: 8GY3). The structure is composed of the catalytic large subunit (L subunit), the small subunit (S subunit), and the cytochrome c subunit (C subunit). In vivo, electrons extracted from acetaldehyde are transferred to ubiquinone via the catalytic center (molybdenum cofactor; Moco) in the L subunit, two iron-sulfur clusters (FeSs) in the S subunit, and three hemes c in the C subunit, in this order. The ET associated with substrate oxidation through the C subunit is conserved in membrane-bound hemoproteins such as alcohol dehydrogenase, fructose dehydrogenase, and glucose dehydrogenase. These enzymes have similarities in high DET activities, and their variants truncating a C subunit, which are advantageous in ignoring interference of detergents and complexity of the multi-step ET through hemes c, have been characterized. However, such research has not been conducted for ALDH, and ALDH has the unique feature of having two cofactors in the S subunit. In this study, we attempted to quantitatively characterize the ALDH variant deleting the C subunit (ΔC_ALDH), focusing on pH dependence of kinetic and thermodynamic parameters.
To construct an expression system of ΔC_ALDH, a plasmid containing genes of L and S subunits of ALDH was transformed into the knock-out strain in which genes of wild-type ALDH were disrupted. ΔC_ALDH was expressed in soluble fraction as expected and successfully purified. Cyclic voltammograms were recorded at ΔC_ALDH-modified multi-walled carbon nanotube-electrodes. The electrode showed clear DET activity, suggesting that the electrode-active site seems to be FeS which is located near the surface of the enzyme. In addition, we investigated the pH dependence of the DET activity and kinetically analyzed the voltammograms using a model in which distribution of enzyme orientation was considered. The kinetic analysis quantitatively estimated the formal potential of the electrode-active site (E°′E), the limiting catalytic current density (j
cat), and the ratio of the maximum value of the standard rate constant of the heterogeneous ET to the catalytic constant in DET-type reaction (k°max/k
c,DET). j
cat exponentially increased from pH 2.5 to 5.5 and reached a plateau between pH 6.0 and 8.0, while E°′E changed by –48 mV pH–1 from pH 2.5 to 5.5 and became constant between pH 6.0 and 8.0. Since the formal potential of acetate/acetaldehyde redox couple (E°′S) changes by –89 mV pH–1, E°′E – E°′S increases as the condition becomes more basic. Then, we focused on a relationship between the potential difference and DET activity. The log (j
cat / mA cm–2) vs. E°′E – E°′S plot showed a linear increasing region and a constant region independent of E°′E – E°′S, as shown in Figure 1. The former slope suggested that DET activity obeyed the ideal linear free energy relationship (LFER) without any specific interaction. In this region, k
c,DET was controlled by the potential difference in the ET pathway. When E°′E – E°′S is sufficiently large, k
c,DET seems to be limited by other factors such as entry of substrate into the catalytic site, a catalytic turnover in Moco, and the intramolecular ET.
On the other hand, in the assay with ferricyanide as an alternative electron acceptor, the slope representing the ideal LFER was not observed. We assume that the potential difference between the enzyme and ferricyanide was insufficient, thus the catalytic constant in solution (k
c,solution) was kinetically analyzed based on the ping-pong bi-bi mechanism considering changes in E°′E, E°′S, and the formal potential of ferricyanide/ferrocyanide. Using parameters obtained from the analysis for j
cat and k
c,solution, Γ
E,eff, k°max, and k
c,DET were separately evaluated with an assumption that Γ
E,eff was independent of pH.
In summary, we quantitively characterized the bioelectrochemical parameters of ΔC_ALDH and compared the properties between DET and ferricyanide reductase activities. This work will be beneficial for understanding ALDH, and comparing it with wild-type ALDH will elucidate the roles of the C subunit.

Figure 1