The Membrane-Bound Quinohemoprotein Alcohol Dehydrogenase from Gluconacetobacter diazotrophicus PAL5 Carries a [2Fe-2S] Cluster

Gómez-Manzo, Saúl; Solano-Peralta, Alejandro; Saucedo-Vázquez, Juan Pablo; Escamilla-Marván, J. Edgardo; Kroneck, Peter M. H.; Sosa-Torres, Martha Elena

The Membrane-Bound Quinohemoprotein Alcohol Dehydrogenase from Gluconacetobacter diazotrophicus PAL5 Carries a [2Fe-2S] Cluster

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Datum

2010

Autor:innen

Gómez-Manzo, Saúl

Solano-Peralta, Alejandro

Saucedo-Vázquez, Juan Pablo

Escamilla-Marván, J. Edgardo

Kroneck, Peter M. H.

Sosa-Torres, Martha Elena

DOI (zitierfähiger Link)

https://doi.org/10.1021/bi9015007

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Biologie

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Zeitschriftenartikel

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Published

Erschienen in

Biochemistry. 2010, 49(11), pp. 2409-2415. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi9015007

Zusammenfassung

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 N₂-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/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.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

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ISO 690

GÓMEZ-MANZO, Saúl, Alejandro SOLANO-PERALTA, Juan Pablo SAUCEDO-VÁZQUEZ, J. Edgardo ESCAMILLA-MARVÁN, Peter M. H. KRONECK, Martha Elena SOSA-TORRES, 2010. The Membrane-Bound Quinohemoprotein Alcohol Dehydrogenase from Gluconacetobacter diazotrophicus PAL5 Carries a [2Fe-2S] Cluster. In: Biochemistry. 2010, 49(11), pp. 2409-2415. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi9015007

BibTex

@article{GomezManzo2010-03-23Membr-37641,
  year={2010},
  doi={10.1021/bi9015007},
  title={The Membrane-Bound Quinohemoprotein Alcohol Dehydrogenase from Gluconacetobacter diazotrophicus PAL5 Carries a [2Fe-2S] Cluster},
  number={11},
  volume={49},
  issn={0006-2960},
  journal={Biochemistry},
  pages={2409--2415},
  author={Gómez-Manzo, Saúl and Solano-Peralta, Alejandro and Saucedo-Vázquez, Juan Pablo and Escamilla-Marván, J. Edgardo and Kroneck, Peter M. H. and Sosa-Torres, Martha Elena}
}

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    <dcterms:abstract xml:lang="eng">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&lt;sub&gt;2&lt;/sub&gt;-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&lt;sub&gt;av&lt;/sub&gt; 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&lt;sub&gt;sq&lt;/sub&gt;). 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&lt;sub&gt;m&lt;/sub&gt; (pH 6.0 vs SHE) of the four c-type cytochromes were estimated to E&lt;sub&gt;m1&lt;/sub&gt; = -64 (+/-2) mV, E&lt;sub&gt;m2&lt;/sub&gt; = -8 (+/-2) mV, E&lt;sub&gt;m3&lt;/sub&gt; = +185 (+/-15) mV, and E&lt;sub&gt;m4&lt;/sub&gt; = +210 (+/-10) mV (spectroelectrochemistry), E&lt;sub&gt;mFeS&lt;/sub&gt; = -250 (+/-5) mV for the [2Fe-2S] cluster, and E&lt;sub&gt;mPQQ&lt;/sub&gt; = -210 (+/-5) mV for the PQQ/PQQH&lt;sub&gt;2&lt;/sub&gt; 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.</dcterms:abstract>
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