Carbon monoxide :acceptor oxidoreductase from Pseudomonas thermocarboxydovorans strain C2 and its use in a carbon monoxide sensor

Abstract

The carbon monoxide:acceptor oxidoreductase from autotrophically-grown Pseudomonas thermocarboxydovorans strain C2 was purified to 95% homogeneity and found to contain fiavin, iron, acid-labile sulphide and possibly molybdenum; its molecular weight was 2.7 × 10 5. The enzyme catalyzed the oxidation of CO to CO 2 with various electron acceptors including phenazine ethosulphate, methylene blue, hexacyanoferrate(III) and ferrocene derivatives (ferrocene monocarboxylic acid, 1,1′dimethylferrocene and horse heart cytochrome C) but not viologen dyes, NAD(P) and oxygen. The optimum pH and temperature for enzyme activity in the in vitro assay were 7.5 and 80°C, respectively. Carbon monoxide is the only known electron donor (apparent K m 5 × 10 −7 M) and acetylene, cyanide, 8-quinolinol and sulphydryl reagents inhibited the enzyme. Second-order homogeneous rate constants for the reaction between the reduced enzyme and either cytochrome C (3.0 × 10 4 l mol −1 s −1) or the ferricinium ion of ferrocene monocarboxylic acid (4.0 × 10 5 l mol −1 s −1) were calculated by using cyclic voltammetry. The latter reaction was exploited by incorporating 1,1′-dimethylferrocene in a carbon electrode and retaining carbon monoxide oxidoreductase behind a membrane at the surface. The probe gave a linear current response to aqueous concentrations of carbon monoxide up to 65 μM and achieved a steady current in < 15 s. A similar configuration was used to detect gaseous carbon monoxide. With a fuel cell mode, 20 nmol could be detected.