The role of blue copper proteins in the oxidation of methylamine by an obligate methylotroph

Abstract

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.

Measurement of the oxidation‐reduction potentials of amicyanin and c‐type cytochromes from Paracoccus denitrificans

“…strain AM1, E0 = 280 mV [8]; Thiobacillus versutus, E0 = 260 mV [9]; and organism 4025, E0 = 294 mV [10]. Cytochrome c-550 is presumably identical to the cytochrome c-550 which was isolated previously from P.…”

Characterization of a novel soluble c-type cytochrome in a moxD mutant of Methylobacterium extorquens AM1

“…Methods for growth and preparation of cell fractions and purified proteins were exactly as described previously : M . methylotrophus (Cross & Anthony, 1980); organism 4025 (Lawton & Anthony, 1985); P . denitriJicans (Beardmore-Gray et al, 1983); A .…”

“…A similar assay was used for testing reaction with methylamine dehydrogenase. The reaction was in 1 ml cuvettes in 20 mM-MOPS buffer (pH 7.0) containing 25 mM-methylamine, 26 pM-cytochrome c-553 and about 0.3 pM-methylamine dehydrogenase, prepared and assayed as previously described (Lawton & Anthony, 1985); its specific activity was 3.5 pmol min-' (mg protein)-' in the standard dye-linked assay.…”

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

“…The copper-containing protein amicyanin is proposed to be the natural electron acceptor for MADH (17,25,34,35,54,55,57,58). Amicyanin is thought to transfer electrons to a soluble periplasmic c-type cytochrome, but some uncertainty exists concerning the identity of the in vivo acceptor.…”

“…Recent evidence has shown that the MADH cofactor has a unique structure, having two tryptophans within the polypeptide chain that are modified and covalently linked (14). A novel structure has been proposed for this cofactor (43).The copper-containing protein amicyanin is proposed to be the natural electron acceptor for MADH (17,25,34,35,54,55,57,58 Another copper-containing protein, azurin, has been found in M. extorquens AML. It accepts electrons in vitro from amicyanin, cytochrome CL, or cytochrome CH and transfers them to a terminal oxidase (54).…”