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Kinetic and structural data are presented for the iron-promoted dehydrogenation of the amine, [Fe(III)L3]3+ (1), L3 = 1,9-bis(2'-pyridyl)-5-[(ethoxy-2''-pyridyl)methyl]-2,5,8-triazanonane. Spectroscopic and electrochemical experiments under the exclusion of dioxygen helped to identify reaction intermediates and the final product, the Fe(II)-monoimine complex [Fe(II)L4]2+ (2), L4 = 1,9-bis(2'-pyridyl)-5-[(ethoxy-2''-pyridyl)methyl]-2,5,8-triazanon-1-ene. 2 is formed by disproportionation of the starting complex 1 by a three-step reaction mechanism, most likely via ligand-centered radical intermediates. The rate law can be described by the second-order rate equation, -d[(Fe(III)L3)3+]/dt = k(EtO)- [(Fe(III)L3)3+][EtO-], with k(EtO)- = 4.92 +/- 0.01 x 104 M(-1) s(-1) (60 degrees C, mu = 0.01 M). The detection of general base catalysis and a primary kinetic isotope effect (k(EtO)-(H)/k(EtO)-(D) = 1.73) represents the first kinetic demonstration that the deprotonation becomes rate determining followed by electron transfer in the oxidative dehydrogenation mechanism. We also isolated the Fe(II)-monoimine complex 2 and determined its structure in solution (NMR) and in the solid state (X-ray).

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.

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