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The membrane-bound quinohemoprotein alcohol. Dehydrogenase from gluconacetobacter diazotrophicus PAL5 carries a [2Fe-2S] Cluster

Abstract:

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 ≊ 72 kDa, subunit II ≊ 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 (gav 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 Em (pH 6.0 vs SHE) of the four c-type cytochromes were estimated, to .Em1 = -64 (±2) m V, E m2 = -8(±2)mV, & Em3 = +185 (±15) mV, and Em4 = +210(±10)mV(spectroelectrochemistry), &E mFeS = -250 (±5) mV for the [2Fe-2S] cluster, and E mPQQ = -210 (±5) mV for the PQQ/PQQH2 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. © 2010 American Chemical Society.