Departamento de Química Inorgánica y Nuclear, Facultad de Química, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria 04510, México, D.F.
Biochemistry. 2010 Mar 23;49(11):2409-15. doi: 10.1021/bi9015007.
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.
谷氨酸醋酸杆菌在醋酸菌中脱颖而出，因为它固定氮气，是一种真正的内生菌。它有一套组成型酶来氧化乙醇和乙醛，这些酶在依赖 N(2)的生长过程中被上调。膜结合的醇脱氢酶 (ADH) 是一种异二聚体（亚基 I 约 72 kDa，亚基 II 约 44 kDa），是该生物体的重要组成部分。Ga. diazotrophicus 的 ADH 是一种典型的醌血蛋白，含有一个吡咯喹啉醌 (PQQ) 和四个 c 型细胞色素。首次通过电子顺磁共振波谱 (EPR) 鉴定了这种酶中的 [2Fe-2S] 簇。这一发现得到了定量化学分析的支持，表明每个 ADH 异二聚体含有 5.90 +/- 0.15 Fe 和 2.06 +/- 0.10 个酸不稳定硫。ADH 的 X 波段 EPR 光谱（在存在二氧的情况下分离，20 K）显示三个宽共振在 g 2.007、1.941 和 1.920（g(av) 1.956），以及一个位于 g = 2.0034 的强烈窄线。后一个信号在 100 K 时仍能检测到，归因于 PQQ 半醌自由基 (PQQ(sq))。在较低温度下观察到的宽共振被分配给一个电子还原状态下的 [2Fe-2S] 簇。四个 c 型细胞色素的氧化还原电位 E(m)（相对于 SHE，pH 6.0）估计为 E(m1) = -64 (+/-2) mV，E(m2) = -8 (+/-2) mV，E(m3) = +185 (+/-15) mV，E(m4) = +210 (+/-10) mV（光谱电化学），[2Fe-2S] 簇的 E(mFeS) = -250 (+/-5) mV，PQQ/PQQH(2) 对的 E(mPQQ) = -210 (+/-5) mV（EPR 光谱）。我们提出了一个模型，用于展示 Ga. diazotrophicus 的膜结合 ADH 内和分子间电子途径的假设。亚基 I 结合 PQQ 辅因子、[2Fe-2S] 簇和一个 c 型细胞色素。亚基 II 含有三个 c 型细胞色素，从而为位于细胞质膜中的醌提供了有效的电子转移途径。
