Purification and characterization of azurin from the methylamine-utilizing obligate methylotroph Methylobacillus flagellatus KT.

Methylamine dehydrogenase (MADH) and azurin were purified from the periplasmic fraction of the methylamine-grown obligate methylotroph Methylobacillus flagellatus KT. The molecular mass of the purified azurin was 16.3 kDa, as measured by SDS-PAGE, or 13 920 Da as determined by MALDI-TOF mass spectrometry. Azurin of M. flagellatus KT contained 1 copper atom per molecule and had an absorption maximum at 620 nm in the oxidized state. The redox potential of azurin measured at pH 7.0 by square-wave voltammetry was +275 mV versus normal hydrogen electrode. MADH reduced azurin in the presence of methylamine, indicating that this cupredoxin is likely to be the physiological electron acceptor for MADH in the electron transport chain of the methylotroph. A scheme of electron transport functioning in M. flagellatus KТ during methylamine oxidation is proposed.

Ferrous iron oxidation in moderately thermophilic acidophile Sulfobacillus sibiricus N1(T).

The iron-oxidizing system of a moderately thermophilic, extremely acidophilic, gram-positive mixotroph, Sulfobacillus sibiricus N1(T), was studied by spectroscopic, high-performance liquid chromatography and inhibitory analyses. Hemes B, A, and O were detected in membranes of S. sibiricus N1(T). It is proposed that the electron transport chain from Fe²(+) to O2 is terminated by 2 physiological oxidases: aa3-type cytochrome, which dominates in the early-exponential phase of growth, and bo3-type cytochrome, whose role in iron oxidation becomes more prominent upon growth of the culture. Both oxidases were sensitive to cyanide and azide. Cytochrome aa3 was more sensitive to cyanide and azide, with K(i) values of 4.1 and 2.5 µmol·L⁻¹, respectively, compared with K(i) values for cytochrome bo3, which were 9.5 µmol·L⁻¹ for cyanide and 7.0 µmol·L⁻¹ for azide. This is the first evidence for the participation of a bo3-type oxidase in ferrous iron oxidation. The respiratory chain of the mixotroph contains, in addition to the 2 terminal oxidases, a membrane-bound cytochrome b573.