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 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.

Purification and Characterization of the Membrane-Bound Quinoprotein Glucose Dehydrogenase of Gluconacetobacter diazotrophicus PAL 5

“…In both cases, a signal was observed at 357 nm, suggesting the presence of PQQ in Ga. diazotrophicus GDHm. The signal at 357 nm is a characteristic signal previously reported in diverse quinoproteins and quinohemoproteins [5,[23][24][25]29]. The presence of PQQ was corroborated spectrofluorometrically of a methanol extract from GDHm (Fig.…”

“…All chemical regents used in this work have been previously described [23,24] and were purchased from SigmaAldrich, SUPELCO-Sigma-Aldrich (St. Louis MO, USA) and Bio-Rad Laboratories (Hercules, CA, USA).…”

“…Then, the cells were washed threefold with 50 mM K 2 HPO 4 , pH 6.5 containing 1 mM MgCl 2 and 1 mM CaCl 2 . Procedures used for cell disruption, and membranes preparations have been described previously [19,23,24]. Membranes were instantly frozen in liquid N 2 in order to preserve and store them.…”

“…After that, the extracted PQQ and the standards quinones were analyzed by RP-HPLC (Waters model 996). The prosthetic group was separated and identified in a RP-analytical column (Waters C18 Spherisorb S5 OD52; 4.6 9 150 mm) as described [24][25][26]. The system was calibrated using the following commercial standards: methanol-extracted PQQ associated with the purified ADHa and ALDH from Ga. diazotrophicus (retention times: 4.5 and 6.9 min, respectively).…”

“…In addition, the PQQ extracted from membrane-bound alcohol dehydrogenase (PQQ-ADHa) was included; PQQ-ADHa contains a PQQ prosthetic group in the form of a semiquinone. The PQQ extracted from aldehyde dehydrogenase (PQQ-ALDH), which is present in the oxidized state [24,25] was also included. The retention time of the PQQ of GDHm was the same as that of the commercial cofactor reduced with sodium dithionite (RT = 4.16 min), represented as PQQH2 in Fig.…”