The membrane-bound quinohemoprotein alcohol dehydrogenase from Gluconacetobacter diazotrophicus PAL5 carries a [2Fe-2S] cluster.
Gluconacetobacter diazotrophicus stands out among the acetic acid bacteria as it fixes dinitrogen and is a true endophyte. It has a set of constitutive enzymes to oxidize ethanol and acetaldehyde which is upregulated during N(2)-dependent growth. The membrane-bound alcohol dehydrogenase (ADH) is a heterodimer (subunit I approximately 72 kDa, subunit II approximately 44 kDa) and constitutes an important component of this organism. ADH of Ga. diazotrophicus is a typical quinohemoprotein with one pyrroloquinoline quinone (PQQ) and four c-type cytochromes. For the first time, a [2Fe-2S] cluster has been identified by EPR spectroscopy in this type of enzyme. This finding is supported by quantitative chemical analysis, revealing 5.90 +/- 0.15 Fe and 2.06 +/- 0.10 acid-labile sulfurs per ADH heterodimer. The X-band EPR spectrum of ADH (as isolated in the presence of dioxygen, 20 K) showed three broad resonances at g 2.007, 1.941, and 1.920 (g(av) 1.956), as well as an intense narrow line centered at g = 2.0034. The latter signal, which was still detected at 100 K, was attributed to the PQQ semiquinone radical (PQQ(sq)). The broad resonances observed at lower temperature were assigned to the [2Fe-2S] cluster in the one-electron reduced state. The oxidation-reduction potentials E(m) (pH 6.0 vs SHE) of the four c-type cytochromes were estimated to E(m1) = -64 (+/-2) mV, E(m2) = -8 (+/-2) mV, E(m3) = +185 (+/-15) mV, and E(m4) = +210 (+/-10) mV (spectroelectrochemistry), E(mFeS) = -250 (+/-5) mV for the [2Fe-2S] cluster, and E(mPQQ) = -210 (+/-5) mV for the PQQ/PQQH(2) couple (EPR spectroscopy). We propose a model for the membrane-bound ADH of Ga. diazotrophicus showing hypothetical intra- and intermolecular electron pathways. Subunit I binds the PQQ cofactor, the [2Fe-2S] cluster, and one c-type cytochrome. Subunit II harbors three c-type cytochromes, thus providing an efficient electron transfer route to quinones located in the cytoplasmic membrane.
Gómez-Manzo
S
S
Departamento de Química Inorgánica y Nuclear, Facultad de Química, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria 04510, México, D.F.
Solano-Peralta
A
A
Saucedo-Vázquez
J P
JP
Escamilla-Marván
J E
JE
Kroneck
P M H
PM
Sosa-Torres
M E
ME
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D003580:Cytochromes / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D041821:Gluconacetobacter / Q000166:cytology* / Q000201:enzymology*; D007501:Iron; D008970:Molecular Weight; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur
D003067:Coenzymes; D003580:Cytochromes; D021122:Protein Subunits; D013455:Sulfur; D007501:Iron; D000426:Alcohol Dehydrogenase