Characterization of a cytochrome a1 that functions as a ubiquinol oxidase in Acetobacter aceti

Abstract

The terminal oxidase for ethanol oxidation in Acetobacter aced was purified as a complex consisting of four subunits (subunits I, II, III, and IV) with molecular masses of 72, 34, 21, and 13 kDa, respectively.Spectrophotometric analysis and catalytic properties determined with the purified enzyme showed that it belonged to a family of cytochrome a, (ba) (cyaB, cyaC, and cyaD) encoding the other three subunits (subunits II, III, and IV) were clustered upstream and downstream of the cyaA gene in the order cyaB, cyat4, cyaC, and cyaD and with the same transcription polarity, forming an operon. As expected from the enzymatic properties, CyaB, CyaC, and CyaD showed great similarity in amino acid sequence to the corresponding subunits of the E. coli o-type ubiquinol oxidase and aa3-tYpe cytochrome c oxidases.The characteristic activity of the genus Acetobacter in vinegar production is the oxidation of ethanol, which is catalyzed by the two membrane-associated dehydrogenases, alcohol dehydrogenase and aldehyde dehydrogenase. The enzymatic properties of these dehydrogenases have been elucidated (1, 2, 9, 34), and the genes encoding these enzymes have been cloned and sequenced (10,32,33). Electrons generated in ethanol oxidation are thought to be directly transferred through the respiratory chain to the terminal oxidase via ubiquinone (20). Acetobacter strains were divided into two groups on the basis of the terminal oxidases; one contains cytochrome a,, and the other contains cytochrome d (3). Williams and Poole (36, 37) observed that the amount of cytochrome o, as the terminal oxidase of Acetobacterpasteurianus NCIB 6428, increased under aerobic culture conditions. Recently, Matsushita et al. (17,19) reported that cytochrome al ofA. aceti, which is composed of four subunits (55,35,22, and 18 kDa) containing heme b, heme a, and one copper ion, functions as a ubiquinol oxidase. They also examined the effect of aeration on the terminal ubiquinol oxidase and observed that a change in culture conditions caused a change in the composition of the terminal ubiquinol oxidase (17, 18).To elucidate the genetic background of the terminal oxidase in ethanol oxidation, we first purified and characterized the terminal oxidase from a thermotolerant strain, A. aceti 1023 (24), a suitable strain for submerged acetic acid fermentation. We then cloned and characterized the gene cluster * Corresponding author.encoding all the subunits of the terminal oxidase. In this paper, we describe the enzymatic properties of the ubiquinol oxidase from A. aceti and the cloning and sequencing of the gene cluster. In addition, we compared the amino acid sequence of each subunit with those of other terminal oxidases, especially the Escherichia coli o-type ubiquinol oxidase, which contains heme b, heme o, and one copper ion (5,22,25).To our knowledge, this is the first report on a gene encoding an a1-type cytochrome functioning as a ubiquinol oxidase.MATERIALS AND METHODS Bacterial strains and plasmids. A. aceti 1023 (24) is an isolate from a vinegar fac...

Probing a Role of Subunit IV of the -type Ubiquinol Oxidase by Deletion and Cross-linking Analyses

“…2). To eliminate Gly 13 to Ala 44 , ⌬D3 was constructed by blunt-end ligation at the NcoI and EagI sites using T4 DNA polymerase and mung bean nuclease, respectively.…”

“…1). CyoD (21) is homologous to subunit IV of ba-type ubiquinol oxidase from A. aceti (13) and P. denitrificans (20), aa 3 -type menaquinol oxidase from B. subtilis (23), and caa 3 -type cytochrome c oxidase from thermophilic Bacillus PS3 (17), B. subtilis (24), and alkalophilic B. firmus OF4 (25). Among these seven subunit IV sequences, 17-53% amino acid residues (32% in average) are conserved, and Phe , and Met 68 in cytoplasmic loop II/III are found to be invariant (Fig.…”

“…Subunits I, II, and III of UQO are encoded by the cyoB, cyoA, and cyoC genes, respectively, of the cyoABCDE operon (19,21,22) and are homologous to the counterparts of mitochondrial cytochrome c oxidase (5). Subunit IV (12 kDa) is conserved in bacterial heme-copper terminal oxidases (13,17,20,21,(23)(24)(25) and is likely to be the cyoD gene product, as demonstrated for the counterpart of ba-type ubiquinol oxidase from Acetobacter aceti (13), aa 3 -type menaquinol oxidase from Bacillus subtilis W23 (14), and caa 3 -type cytochrome c oxidase from thermophilic Bacillus PS3 (17). Subunit IV of UQO, which consists of three putative transmembrane helices (26), however, is unlikely to be a homologue of either nuclear-encoded small subunits of mitochondrial cytochrome c oxidase (21) or subunit IV of aa 3 -type cytochrome c oxidase from Paracoccus denitrificans that has a single transmembrane helix and resides in a cleft between subunits I and III (15,16 (18,19) is not encoded by the cyoE gene (29).…”

“…Bacterial heme-copper terminal quinol (10,13,14) and cytochrome c (15)(16)(17) oxidases generally consist of four different subunits, I to IV. In some preparations, the fifth polypeptide with a molecular mass of 28 kDa was associated with the oxidase complex (18 -20).…”

Quinoprotein Alcohol Dehydrogenase of Acetic Acid Bacteria: Kinetic Study on the Enzyme Purified fromAcetobacter methanolicus

“…3 ,4) The ethanol oxidase respiratory chain has been studied in several acetic acid bacteria and shown to consist of ADH, ubiquinone, and either cytochrome ba or cytochrome bo ubiquinol oxidase. 3 ,4.15 18) In this work we describe a more effective new purification procedure for ADH from A. methanolicus. Kinetic properties of this ADH using artificial electron acceptors such as potassium ferricyanide, DCIP, PMS, and ubiqu.lnones-l (QI) and 2 (Q2) were investigated.…”