The nitric oxide production in the moss Physcomitrella patens is mediated by nitrate reductase.

During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.

The role of molecular oxygen in the iron(III)-promoted oxidative dehydrogenation of amines.

A mechanistic study is presented of the oxidative dehydrogenation of the iron(III) complex [Fe(III)L(3)](3+), 1, (L(3) = 1,9-bis(2'-pyridyl)-5-[(ethoxy-2''-pyridyl)methyl]-2,5,8-triazanonane) in ethanol in the presence of molecular oxygen. The product of the reaction was identified by NMR spectroscopy and X-ray crystallography as the identical monoimine complex [Fe(II)L(4)](2+), 2, (L(4) = 1,9-bis(2'-pyridyl)-5-[(ethoxy-2''-pyridyl)methyl]-2,5,8-triazanon-1-ene) also formed under an inert nitrogen atmosphere. Molecular oxygen is an active player in the oxidative dehydrogenation of iron(III) complex 1. Reduced oxygen species, e.g., superoxide, (O2˙(-)) and peroxide (O2(2-)), are formed and undergo single electron transfer reactions with ligand-based radical intermediates. The experimental rate law can be described by the third order rate equation, -d[(Fe(III)L(3))(3+)]/dt = kOD[(Fe(III)L(3))(3+)][EtO(-)][O2], with kOD = 3.80 ± 0.09 × 10(7) M(-2) s(-1) (60 °C, µ = 0.01 M). The reduction O2 → O2˙(-) represents the rate determining step, with superoxide becoming further reduced to peroxide as shown by a coupled heme catalase assay. In an independent study, with H2O2, replacing O2 as the oxidant, the experimental rate law depended on [H2O2]: -d[(Fe(III)L(3))(3+)]/dt = kH2O2[(Fe(III)L(3))(3+)][H2O2]), with kH2O2 = 6.25 ± 0.02 × 10(-3) M(-1) s(-1). In contrast to the reaction performed under N2, no kinetic isotope effect (KIE) or general base catalysis was found for the reaction of iron(III) complex 1 with O2. Under N2, two consecutive one-electron oxidation steps of the ligand coupled to proton removal produced the iron(II)-monoimine complex [Fe(II)L(4)](2+) and the iron(II)-amine complex [Fe(II)L(3)](2+) in a 1 : 1 ratio (disproportionation), with the amine deprotonation being the rate determining step. Notably, the reaction is almost one order of magnitude faster in the presence of O2, with kEtO(-) = 3.02 ± 0.09 × 10(5) M(-1) s(-1) (O2) compared to kEtO(-) = 4.92 ± 0.01 × 10(4) M(-1) s(-1) (N2), documenting the role of molecular oxygen in the dehydrogenation reaction.

The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

The active (ADHa) and inactive (ADHi) forms of the PQQ-alcohol dehydrogenase from Gluconacetobacter diazotrophicus differ in their respective oligomeric structures and redox state of their corresponding prosthetic groups.

The membrane-bound alcohol dehydrogenase of Gluconacetobacter diazotrophicus contains one pyrroloquinoline quinone moiety (PQQ), one [2Fe-2S] cluster, and four c-type cytochromes. Here, we describe a novel and inactive enzyme. ADHi, similarly to ADHa, is a heterodimer of 72- and 44-kDa subunits and contains the expected prosthetic groups. However, ADHa showed a threefold molecular mass as compared to ADHi. Noteworthy, the PQQ, the [2Fe-2S] and most of the cytochromes in purified ADHi is in the oxidized form, contrasting with ADHa where the PQQ-semiquinone is detected and the [2Fe-2S] cluster as well as the cytochromes c remained fully reduced after purification. Reduction kinetics of the ferricyanide-oxidized enzymes showed that while ADHa was brought back by ethanol to its full reduction state, in ADHi, only one-quarter of the total heme c was reduced. The dithionite-reduced ADHi was largely oxidized by ubiquinone-2, thus indicating that intramolecular electron transfer is not impaired in ADHi. The acidic pH of the medium might be deleterious for the membrane-bound ADH by causing conformational changes leading to changes in the relative orientation of heme groups and shift of corresponding redox potential to higher values. This would hamper electron transfer resulting in the low activity observed in ADHi.

On the mechanism of iron(III)-dependent oxidative dehydrogenation of amines.

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).