SEQUENCE OF ELECTRON CARRIERS IN THE PROCESS OF METHANOL OXIDATION BYA NEW OBLIGATE METHYLOTROPHIC BACTERIUM

From pink soluble fractions prepared from cells cultured in a copper-f ree medium, active methanol dehydrogenase (MDH) and two soluble c-type cytochromes (c-I and c-II) were purified homogeneously. The green fra ctions from cells grown on a medium containing 1.0 mg/l of copper had inactive MDH, cytochrome c-II, and blue copper protein. The amount of copper retained in the blue copper protein increased with cultivation time. The oxidized blue copper protein was similar to the classical ty pe I blue copper proteins since it had the novel absorption peak at 62 5 nm. However, when the blue protein was reduced with MDH or dithionit e, it showed the same spectrum as ferrocytochrome c-I. The isoelectric points of cytochrome c-I, blue copper protein and cytochrome c-II wer e 9.08, 9.08 and 6.52, respectively. These results suggest that the id entity of the purified blue copper protein is cytochrome c-I, and copp er ions bind to the cytochrome as methanol is depleted in the culture medium. In addition, MDH activity was not detected at all in the metha nol-depleted condition. The data suggest that blue copper protein acts as a negative regulator for MDH, The electrons were transferred as fo llows: MDH --> cytochrome c-II --> cytochrome c-I (blue copper protein ). It was also revealed that the initial 'docking' of MDH and cytochro me c-II is accompanied by electrostatic interactions between lysine or arginine residues on the alpha-subunit of MDH and carboxyl groups on the cytochrome c-II.