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 approximate to 72 kDa, subunit II approximate to 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 g2.007, 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.