Transport limited adsorption experiments give a new lower estimate of the turnover frequency of Escherichia coli hydrogenase 1

• Published in: BBA advances Vol. 3; p. 100090
• Main Authors: Aldinio-Colbachini, Anna, Fasano, Andrea, Guendon, Chloé, Jacq-Bailly, Aurore, Wozniak, Jérémy, Baffert, Carole, Kpebe, Arlette, Léger, Christophe, Brugna, Myriam, Fourmond, Vincent
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2023; Elsevier
• Subjects: Biochemistry; Biochemistry, Molecular Biology; Bioinorganic chemistry; Chemical Sciences; Hydrogenase; Life Sciences; Metalloenzyme; Other; Protein film electrochemistry; Hydrogenase; Protein film electrochemistry; Metalloenzyme; Bioinorganic chemistry
• ISSN: 2667-1603; 2667-1603
• DOI: 10.1016/j.bbadva.2023.100090

•E. coli hydrogenase 1 adsorbs onto graphite electrodes even from sub-nanomolar solutions.•this process is mass-transport limited.•this process allows one to provide a lower limit for the value of kcat for the enzyme, which is much higher than that determined from solution assays. Protein Film Electrochemistry is a technique in which a redox enzyme is directly wired to an electrode, which substitutes for the natural redox partner. In this technique, the electrical current flowing through the electrode is proportional to the catalytic activity of the enzyme. However, in most cases, the amount of enzyme molecules contributing to the current is unknown and the absolute turnover frequency cannot be determined. Here, we observe the formation of electrocatalytically active films of E. coli hydrogenase 1 by rotating an electrode in a sub-nanomolar solution of enzyme. This process is slow, and we show that it is mass-transport limited. Measuring the rate of the immobilization allows the determination of an estimation of the turnover rate of the enzyme, which appears to be much greater than that deduced from solution assays under the same conditions.