Functional Expression of a Mo-Cu-Dependent Carbon Monoxide Dehydrogenase (CODH) and Its Use as a Dissolved CO...

Herein, we report the heterologous expression in Escherichia coli of a Mo-Cu-containing carbon monoxide dehydrogenase (Mo-Cu CODH) from Hydrogenophaga pseudoflava, which resulted in an active protein catalyzing CO oxidation to CO₂. By supplying the E. coli growth medium with Na₂MoO₄ (Mo) and CuSO₄ (Cu), the Mo-Cu CODH metal cofactors precursors, the expressed L-subunit was found to have CO-oxidation activity even without the M- and S- subunits. This successful expression of CO-oxidizing-capable single L-subunit provides direct evidence of its role as the catalytic center of Mo-Cu CODH that has not been discovered and studied before. Subsequently, we used the expressed protein to construct a CO bio-microsensor based on a newly developed fast and sensitive Clark-type CO₂ transducer using an aprotic solvent/ionic liquid electrolyte. The CO bio-microsensor exhibited a linear response to CO concentration in the 0-9 μM range, with a limit of detection (LOD) of 15 nM CO. The sensor uses a mixture of Mo-Cu CODH's L-subunit/Mo, Cu cofactors/methylene blue, confined in the enzyme chamber that is placed in front of a CO₂ transducer. The optimized sensor's sensitivity and performance were retained to levels of at least 80% for 1 week of continuous polarization and operation in an aqueous medium. We have also demonstrated the use of an alkaline front-trap solution to make a completely O₂/CO₂ interference-free microsensor. The CO bio-microsensor developed in this study is potentially useful as an analytical tool for the detection of trace CO in dissolved form for monitoring dissolved CO concentration dynamics in natural or synthetic systems.