Organism 4025, an obligate methylotroph, when grown on methylamine in the presence of a high concentration of copper, contained high concentrations of methylamine dehydrogenase and two blue copper proteins, amicyanin and an azurintype protein; these were purified to homogeneity and characterized. The methylamine dehydrogenase is a basic protein (pI8.8) and consists of light and heavy subunits (Mr 14100 and 43000; total Mr 112000). This dehydrogenase differed slightly from other methylamine dehydrogenases in its absorption spectrum and in its lack of thermal stability. Amicyanin, the more abundant blue copper protein, had an Mr of 11 500, a midpoint redox potential of 294mV at pH 7.0, and a much lower isoelectric point (pl 5.3) than other amicyanins. Its absorption maximum was 620nm (7-24nm higher than those of other amicyanins); its absorption coefficient (at 620nm) was 3.8 mm-'1 cm-'. The 'azurin' (6% of the blue copper protein) had an Mr of 12 500, a midpoint redox potential of 323 mV and a high isoelectric point (pl 9.4). Its absorption maximum was 620 nm, the absorption coefficient (16mM-1 cm-') at this wavelength being considerably greater than that of any blue copper protein described previously. The partially-purified soluble cytochromes CH and CL were similar to those of other methylotrophs. The interactions of the purified redox proteins were investigated in order to elucidate their role in methylamine oxidation. Methylamine dehydrogenase was able to donate electrons only to amicyanin, the rate of reaction being 2.04mmol/min per jumol of methylamine dehydrogenase; this is sufficient to account for the rate of respiration in whole bacteria. The blue copper proteins were able to react rapidly with each other and with both the soluble cytochromes c.

Organism 4025 contained only b-type and c-type cytochromes. In the absence of any a-type cytochrome oxidase it was concluded that respiration in these bacteria is catalysed entirely by way of the o-type cytochrome oxidase. After growth on methanol, the soluble protein contained cytochromes cH and cL, and a single blue copper protein of unknown function, 'azurin'. After growth on methylamine the soluble proteins were strikingly different. The total concentration of soluble cytochrome c was about 70% lower; cytochrome cL was about 65%, and cytochrome cH about 16% of the concentrations present after growth on methanol. Although some 'azurin' was still present, a second copper protein, amicyanin (94% of the total), was induced during growth on methylamine. The concentration of this protein was considerably higher than that of blue copper proteins measured in other bacteria and 35 times higher than the concentration in the facultative methylotroph Pseudomonas AM1 during growth on methylamine. It is the very high concentration of amicyanin that gave suspensions of methylamine-grown organism 4025 their blue-green colour. All of the soluble cytochrome c, and all of the methanol dehydrogenase, methylamine dehydrogenase and blue copper proteins were located in the periplasmic fraction. All these results are consistent with the conclusion that the main electron acceptor for methylamine dehydrogenase in organism 4025 is amicyanin.