Attempt to purify and identify an unknown cytochrome c in Ideonella dechloratans

Abstract:

Oxochlorates are compounds consisting of chlorine and oxygen and are in high concentrations toxic to mammals and the environment. They exist and can be formed naturally but most of the oxochlorates in the environment are a result of human activities, for example the paper industry. Bacteria capable of growth anaerobically with (per)chlorate as terminal electron acceptor in its respiratory chain are utilized to purify the waste water. One of those bacteria is Ideonella dechloratans, which uses chlorate reductase to reduce chlorate into chlorite and then chlorite dismutase to decompose chlorite into chlorine and molecular oxygen.

Present work deals with detection and purification of c cytochromes in I. dechloratans that could have a role in electron transport during chlorate reduction. Earlier work has demonstrated the presence of two major periplasmic c cytochromes with apparent molecular weights around 6 and 13 kDa. These have been purified and characterized. The "6 kDa protein" was shown to be capable of donating electrons to chlorate reductase and terminal oxidase in vitro. The molecular weight as determined by mass spectrometry analysis was shown to be 9.4 kDa. The "13 kDa protein" was shown to be unable to act as an electron donor directly to chlorate reductase in vitro. Attempts with mass spectrometry analysis has yet been unsuccessful.

In this study, attempts were made to purify this 13 kDa cytochrome c from anaerobically cultivated I. dechloratans to investigate its properties with 2D electrophoresis and if possible, cut out a spot and analyze it with mass spectrometry.

Some purification was achieved with cationic exchange chromatography, where about 10 other proteins were eluted at the same time as the 13 kDa cytochrome c. Further purification attempts had difficulties with the detection of the cytochrome c on SDS-PAGE. It is less likely, but still possible, that this was due to protein degradation or loss of the heme group. Fractions containing the 13 kDa cytochrome c were analyzed with 2D electrophoresis but the protein could not be detected on the gel. This is probably due to the protein having difficulties entering the IPG strip during the rehydration in the first dimension or difficulties transferring from the strip into the gel in the second dimension. Different changes, such as a low voltage applied over the strip during rehydration and increased carrier ampholyte concentration, were made to the protocol but did not help. Attempts to verify if the problem is in the first or second dimension were made but did not show anything.