Properties and electron transfer specificity of copper proteins from the denitrifier "Achromobacter cycloclastes"

Abstract

A blue copper protein (Mr 12,000) was purified from cells of "Achromobacter cycloclastes" grown as a denitrifier. When reduced, the blue copper protein transferred electrons to the copper protein nitrite reductase purified from the same cells, whereas a variety of cytochromes from denitrifiers failed to do so. Inclusion of a protease inhibitor, phenylmethylsulfonyl fluoride, in the buffers employed during preparation yielded purified blue copper protein with 18 more amino acid residues and two times more specific enzyme activity than other researchers have found.

The purification of a cd1‐type nitrite reductase from, and the absence of a copper‐type nitrite reductase from, the aerobic denitrifier Thiosphaera pantotropha; the role of pseudoazurin as an electron donor

“…On reduction with dithionite the protein is colourless and the visible absorbances disappear. The spectra are very similar to those of pseudoazurins from Alcaligenes faecalis S-6 (Kakutani et al, 1981 b), Methylobacterium extorquens AM1 (formerly Pseudomonas M I ) (Tobari, 1984) and also Achromobacter cycloclastes (Liu et al, 1986) and azurin from P. denitrificans (Martinkus et al, 1980). The molecular mass of pseudoazurin from T. pantotropha was determined by electrospray mass spectrometry of the purified protein and was found to be 13 344 2 0.5 Da.…”

“…Pseudoazurins have previously only been implicated as electron donors to the copper-type nitrite reductase (Kakutani et al, 1981b andLiu et al, 1986), but the pseudoazurin purified here can donate electrons to purified cd,-type nitrite reductase. Azurin from Ps.…”

“…Azurin has a visible spectrum with a single absorbance band under oxidised conditions with a maximum around 620 nm (Adman, 1985). Pseudoazurin has a spectrum with absorbance peaks around 450 nm, 590 nm and 750 nm in the oxidised form (Kakutani et al, 1981b;Tobari, 1984 andLiu et al, 1986). Azurin purified from P. denitrijicans is more similar to pseudoazurin than azurin spectroscopically, and Ambler and Tobari (1985) hence suggested it to be a pseudoazurin.…”

Type 1, blue copper proteins constitute a respiratory nitrite‐reducing system in Pseudomonas aureofaciens

“…The Pseudomonas enzyme is an acidic protein (PI 6.05), whereas the Achromobacter protein is basic (PI 8.3); the former enzyme reacts with a blue copper protein as electron donor and does not utilize oxygen, whereas the latter reacts with cytochrome c-553 and has oxidase activity. Blue copper proteins were previously found to be electron donors for the copper nitrite reductases from Alcaligenes faecalis S-6 [37] and 'Achromobacter cycloclastes' [38]. The enzymes from those organisms, however, are spectroscopically clearly distinct from the Pseudomonas nitrite reductase.…”

The electron transfer complex between nitrous oxide reductase and its electron donors

“…A structural model for the electron transfer complex between P. denitrificans N 2 OR and either P. panthotropus cytochrome c-550 or P. panthotropus pseudoazurin has been proposed on the basis of a theoretical docking study [23]. In the case of A. cycloclastes N 2 OR, its electron donor was shown to be only pseudoazurin [24], since no small cytochrome c was identified in the periplasm of the bacteria growing under denitrifying conditions [25]. Nevertheless, it was shown that bovine heart cytochrome c was also able to reduce the CuA center [26].…”