Abstract
Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb3-type oxidases show 12 conserved histidines. Six of them are diagnostic of heme-copper oxidases and are thought to bind the following cofactors: the low spin heme B and the binuclear high spin heme B-CuB center. The other six are FixN(CcoN)-specific and their function is unknown. To analyze the contribution of these 12 invariant histidines of FixN in cofactor binding and function of the Bradyrhizobium japonicum cbb3-type oxidase, they were substituted by valine or alanine by site-directed mutagenesis. The H131A mutant enzyme had already been reported previously to be defective in oxidase assembly and function (Zufferey, R., Th¿ny-Meyer, L., and Hennecke, H. (1996) FEBS Lett. 394, 349-352). Four of the remaining histidines were not essential for activity or assembly (positions 226, 246, 333, and 457); by contrast, histidines 331, 410, and 418 were required both for activity and stability of the enzyme. The last group of mutant enzymes, H420A, H280A, H330A, and H316V, were assembled but not functional. To purify the latter mutant proteins and the wild-type enzyme, a six-histidine tag was added to the C terminus of subunit I. The His6-tagged cbb3-oxidase complexes were purified 20-fold by a three-step purification protocol. With the exception of the H420A mutant oxidase, the mutant enzymes H280A, H316V, and H331A contained normal amounts of copper and heme B, and they displayed similar visible light spectroscopic characteristics like the wild-type His6-tagged enzyme. The His6-tagged H420A mutant oxidase differed from the His6-tagged wild-type protein by showing altered visible light spectroscopic characteristics. No stable mutant oxidase lacking copper or heme B was obtained. This strongly suggests that copper and heme B incorporations in subunit I are prerequisites for assembly of the enzyme.
Highlights
Terminal cytochrome c oxidases are multimeric membrane protein complexes that catalyze the 2-electron oxidation of cytochrome c and the 4-electron reduction of oxygen to water
Cytochrome c oxidases belong to the so-called superfamily of heme-copper oxidases. They have in common a low spin heme moiety and a so-called binuclear center composed of a high spin heme and
Other typical features of cytochrome c oxidases are the presence of (i) a binuclear CuA center in subunit II, which is the entry site for electrons derived from reduced cytochrome c, and (ii) a Mn2ϩ or Mg2ϩ redox-inactive center that lies between subunits I and II [6, 7]
Summary
Purification of the His6-tagged cbb3-Oxidase from Strain Bj4621— Membrane fractions of anaerobically grown 10-liter batch cultures were isolated as described previously [17] with the following modifications: the membranes were solubilized (45 min, 4 °C) in 20 mM Tris-HCl (pH 8.0), 1 mM phenylmethanesulfonyl fluoride, 100 mM NaCl, and 0.5% dodecyl maltoside (Sigma) at a protein concentration of approximately 3 mg/ml. The cbb3-oxidase was eluted with 3 volumes of buffer A in which the NaCl concentration was 250 mM To this fraction was added 5 mM imidazole before loading it on a Ni2ϩ affinity column (His Bond Resin, Novagen) that had been prepared as described. The copper content of the Chelex 100-treated, purified cytochrome c oxidase was determined by Flame Atomic Absorption Spectrometry (Perkin-Elmer, model 5000)
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