Carbon monoxide dehydrogenase from Rhodospirillum rubrum

Abstract

The carbon monoxide dehydrogenase from the photosynthetic bacterium Rhodospirillum rubrum was purified over 600-fold by DEAE-cellulose chromatography, heat treatment, hydroxylapatite chromatography, and preparative scale gel electrophoresis. In vitro, this enzyme catalyzed a two-electron oxidation of CO to form CO2 as the product. The reaction was dependent on the addition of an electron acceptor. The enzyme was oxygen labile, heat stable, and resistant to tryptic and chymotryptic digestion. Optimum in vitro activity occurred at pH 10.0. A sensitive, hemoglobin-based assay for measuring dissolved CO levels is presented. The in vitro Km for CO was determined to be 110 ,uM. CO, through an unknown mechanism, stimulated hydrogen evolution in whole cells, suggesting the presence of a reversible hydrogenase in R. rubrum which is CO insensitive in vivo.

A “Radical Dance” in Thiamin Biosynthesis: Mechanistic Analysis of the Bacterial Hydroxymethylpyrimidine Phosphate Synthase

“…Generation of formaldehyde during the ThiC‐catalyzed reaction was not detected by either method. To test for carbon monoxide production, an anaerobic carbon monoxide (CO) trapping assay,19 which involves a specific change in the Soret‐region absorbance of hemoglobin (Figure 1 C) as a result of its association with CO to form carboxyhemoglobin, was adapted. With this assay, the formation of CO during the ThiC‐catalyzed conversion of AIR to HMP‐P was clearly detected.…”

Growth of Rhodospirillum rubrum on synthesis gas: Conversion of CO to H2 and poly‐β‐hydroxyalkanoate

“…Two 110 mL samples were collected every 24 h with the first vial serving as a liquid waste bottle to purge the sample line. The optical density at 680 nm (OD 680 nm ), protein concentration, dry weight, and dissolved CO were determined by the methods of Bonam et al (1984); Koch (1994); and Lowry et al (1951), respectively. Liquid samples for dissolved CO analysis were determined using the continuous flow-syringe sampling port described by Kapic (2005).…”

“…R. rubrum can utilize CO under anaerobic conditions as a sole carbon and energy source in the presence or absence of light (Kerby et al, 1992(Kerby et al, , 1995. When exposed to CO, both a carbon monoxide dehydrogenase and a CO-insensitive hydrogenase are induced (Bonam et al, 1989(Bonam et al, , 1984Ensign and Ludden, 1991). The combined activities of these two enzymes result in the water-gas shift reaction: CO þ H 2 O !…”

Nickel utilization by microorganisms.

“…CO dehydrogenase has been partially characterized from two purple, nonsulfur phototrophs. The enzyme was purified 600-fold from Rhodospirillum rubrum grown under anaerobic, phototrophic conditions (31). The cells were found to take up 63Ni very poorly from the medium; however, trace amounts of the radioisotrope did comigrate with the enzyme on native gels.…”