ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P_i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F₁-ATPase from beef heart mitochondria (ndMF₁) has been investigated. It is shown for the first time that P_i decreases the apparent rate constant of uni-site ATP binding by ndMF₁ 3-fold with the K_d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P_i also shifts equilibrium between bound ATP and ADP + P_i in the direction of ATP synthesis with the K_d of 0.17 ± 0.03 mM. However, 10 mM P_i does not significantly affect ATP binding during multi-site catalysis.     

Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase 1 from Methicillin-Resistant Staphylococcus aureus MRSA252 Provides Novel Insights into Substrate Binding and Catalytic Mechanism

The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD⁺. A new phosphate recognition site, “new P_i” site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 Å away from the “classical P_i” site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S + H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the “P_s” site in the ternary complexes but at the “new P_i” site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the “new P_i” site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the “flip-flop” model proposed for the enzyme mechanism.     

Oxidative addition of 3,6-di-tert-butyl-o-benzoquinone and 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone to SnCl2

Addition of 3,6-di-tert-butyl-o-benzoquinone (3,6-DBBQ) to SnCl₂ in THF leads to the oxidation of Sn(II) to Sn(IV) with formation of catecholate complex (3,6-DBCat)SnCl₂ · 2THF (1), where 3,6-DBCat is 3,6-di-tert-butyl-catecholate dianion. The reaction of 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone (IBQ-Prⁱ) also proceeds on the oxidative-addition mechanism yielding bis-iminosemiquinonato species (ISQ-Prⁱ)₂SnCl₂(2), where ISQ-Prⁱ is anion-radical 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzosemiquinolate. The complexes have been characterized by IR, X-band EPR, ¹H NMR (for 1) spectroscopy and magnetochemistry (for 2). X-ray analysis data show the distorted octahedral environment of tin(IV) for both complexes. Complex 1 is diamagnetic (ground state S = 0), while 2 has triplet ground state (S = 1, biradical). Catecholate complex 1 is able to be a spin trap for different organic radicals.     

Design of novel iron compounds as potential therapeutic agents against tuberculosis

In the search for new therapeutic tools against tuberculosis two novel iron complexes, [Fe(L-H)₃], with 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives (L) as ligands, were synthesized, characterized by a combination of techniques, and in vitro evaluated. Results were compared with those previously reported for two analogous iron complexes of other ligands of the same family of quinoxaline derivatives. In addition, the complexes were studied by cyclic voltammetry and EPR spectroscopy. Cyclic voltammograms of the iron compounds showed several cathodic processes which were attributed to the reduction of the metal center (Fe(III)/Fe(II)) and the coordinated ligand. EPR signals were characteristic of magnetically isolated high-spin Fe(III) in a rhombic environment and arise from transitions between m_S = ± 1/2 (g_eff ~ 9) or m_S = ± 3/2 (g_eff ~ 4.3) states. Mössbauer experiments showed hyperfine parameters that are typical of high-spin Fe(III) ions in a not too distorted environment. The novel complexes showed in vitro growth inhibitory activity on Mycobacterium tuberculosis H₃₇Rv (ATCC 27294), together with very low unspecific cytotoxicity on eukaryotic cells (cultured murine cell line J774). Both complexes showed higher inhibitory effects on M. tuberculosis than the “second-line” therapeutic drugs.     

Morphological and physiological adaptive traits of Mediterranean narrow endemic plants: The case of Centaurea gymnocarpa (Capraia Island,Italy)

A case study on Centaurea gymnocarpa Moris & De Not., a narrow endemic species, was carried out by analyzing its morphological, anatomical, and physiological traits in response to natural habitat stress factors under Mediterranean climate conditions. The results underline that the species is particularly adapted to the environment where it naturally grows. At the plant level, the above-ground/below-ground dry mass (1.73 ± 0.60) shows its investment predominately in the above-ground structure with a resulting total leaf area per plant of 1399 ± 94 cm². The senescent attached leaves at the base of the plant contribute to limit leaf transpiration by shading soil around the plant. Moreover, the dense C. gymnocarpa leaf pubescence, leaf rolling, the relatively high leaf mass area (LMA = 12.3 ± 1.3 mg cm⁻²) and leaf tissue density (LTD = 427 ± 44 mg cm⁻³) contribute to limit leaf transpiration, also postponing leaf death under dry conditions. At the physiological level, a relatively low respiration/photosynthesis ratio (R/P_N) in spring results from high R [2.26 ± 0.59 μmol (CO₂) m⁻² s⁻¹] and P_N [12.3 ± 1.5 μmol (CO₂) m⁻² s⁻¹]. The high photosynthetic nitrogen use efficiency [PNUE = 15.5 ± 0.4 μmol (CO₂) g⁻¹ (N) s⁻¹] shows the large amount of nitrogen (N) invested in the photosynthetic machinery of new leaves, associated to a high chlorophyll content (Chl = 35 ± 5 SPAD units). On the contrary, the highest R/P_N ratio (1.75 ± 0.19) in summer is due to a significant P_N decrease and increase of R in response to drought. The low PNUE [1.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] in this season is indicative of a greater N investment in leaf cell walls which may contribute to limit transpiration. On the contrary, the low R/P_N ratio (0.05 ± 0.02) in winter is resulting from the limited enzyme activity of the respiratory apparatus [R = 0.23 ± 0.08 μmol (CO₂) m⁻² s⁻¹] while the low PNUE [3.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] suggests that low temperatures additionally limit plant production. The experiment of the imposed water stress confirms that the C. gymnocarpa growth capability is in conformity with the severe conditions of its natural habitat, likewise as it may be the case with others narrow endemic species that have occupied niches with similar extreme conditions.     

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D_phys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R_g) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R_g value extrapolated from kinetic R_g data to time zero, R_g,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R_g² was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R_g of the minor component of D_phys containing the non-native cis proline isomer (D_phys,cis) to be 25.7 ± 0.6 Å. Moreover, R_g of the major component of D_phys containing the native proline isomer (D_phys,tra) was estimated as 23.9 ± 0.2 Å based on R_g,0. Consequently, both components of the burst-phase intermediate of barnase (D_phys,tra and D_phys,cis) are still largely expanded. It was inferred that D_phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.     

Chemical implantation of Group 4 cations on silica via cyclopentadienyl- and N,N-dialkylcarbamato derivatives

Chemical implantation of Group 4 cations [Ti(III), Ti(IV), Zr(IV), Hf(IV)] has been carried out under mild conditions by the reaction of polycyclopentadienyl- (MCp_n; M = Ti, n = 3, 4; M = Zr, Hf, n = 4), mixed cyclopentadienyl/N,N-dialkylcarbamato (ML_x(O₂CNEt₂)_y; M = Ti, L = Cp, C₅Me₅ (Cp*), x = 2, y = 1; M = Hf, L = Cp, x = 1, y = 3), and N,N-dialkylcarbamato (M(O₂CNR₂)_n, M = Ti, n = 3, R = ⁱPr; M = Ti, Hf, n = 4, R = Et; M = Zr, n = 4, R = ⁱPr) derivatives, with the silanol groups of amorphous silica. Cyclopentadiene/pentamethylcyclopentadiene and/or carbon dioxide and the secondary amine are released in the process. The amount of implanted cations depends on the metal and on the ligands, the pentamethylcyclopentadienyl complex being less reactive than the unsubstituted congener. The starting complexes and the final products have been characterized by EPR or by ¹³C CP-MAS NMR spectroscopy.     

Effect of substituent dtp to optical properties of heterobimetallic M/Ag/S nest-shaped clusters (M = Mo, W)

Clusters [MoS₄Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (1), [WS₄Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (2) and [WOS₃Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (3) were synthesized by the reaction of (NH₄)₂MoS₄/(NH₄)₂WS₄, (NH₄)₂WOS₃ with Ag[S₂P(OPrⁱ)₂]. Their structures have been characterized by X-ray diffraction. The clusters consist of a distorted tetrahedral MS₄ (or MOS₃) (M = Mo, W) with three Ag atoms and three sulfur atom bridges (Fig. 1), and resemble roughly that of cubane-like clusters. The nonlinear optical (NLO) properties were studied with an 8 ns pulsed laser at 532 nm. Its optical response to the incident light exhibits good optical absorptive and refractive effects, with α₂ = 1.56 × 10⁻¹⁰ m W⁻¹, n₂ = 3.87 × 10⁻¹⁷ m² W⁻¹ for cluster 1; α₂ = 1.33 × 10⁻¹⁰ m W⁻¹, n₂ = 6.52 × 10⁻¹⁷ m² W⁻¹for cluster 2; and α₂ = 2.54 × 10⁻¹⁰ m W⁻¹, n₂ = 4.07 × 10⁻¹⁷ m² W⁻¹ for cluster 3 for a 1.56 × 10⁻⁴ mol dm⁻³ CH₂Cl₂ solution.     

Inter-annual variability of Potamogeton perfoliatus stands

Yearly differences in the composition and nutrient content of submerged macrophyte stands, mainly of the long-term dominant Potamogeton perfoliatus L. were observed at 10 stations along 83 km route of the Estonian shore of L. Peipsi (total area 3555 km²) in the period of 1999–2002. Changes consisted in the differences in the presence of plants (three stations), replacement of dominant (one station), removal of mud sediment (one station), mass propagation of Lemna trisulca (all stations) in 1999 and of Cladophora glomerata on P. perfoliatus (six stations) in 2000. The amount of C. glomerata on pondweed, similar to the biomass of host plant, decreased in 2001–2002, whereas markedly thick layer of epiphytic microalgae covered P. perfoliatus in 2002. In the samples of P. perfoliatus collected at the stations of the mass propagation of C. glomerata in the next year, 2001, inflorescences were absent. The appearance of an increase in P. perfoliatus stands was probably associated with improving sediment conditions in the period of low water level in the last observation years. A trend of constant increase in the mean molar N/P ratio in the tissues of P. perfoliatus in the study period (from 17.8 to 25.2) was statistically significant (p = 0.003). Classical canonical analysis, used to study two complexes correlated between themselves characteristics of P. perfoliatus and environmental conditions, revealed that plant growth factor (including biomass, tissue N%, P% and N/P) was in strong correlation (p < 0.0001) with water condition factor (including mean temperature in May, sum of temperatures between ice break and sampling, mean water level, N_tot and P_tot of water in June). More important for water condition factor were mean temperature in May and P_tot of water in June. Also the independent significance of water N_tot and P_tot for pondweeds was tested and confirmed by ANOVA analysis.     

Manganese(III) and rhenium(II) complexes with bulky 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinonato ligands via carbonyls of corresponding metals

Four-coordinate complex Mn^III(ISQ-Prⁱ)(AP-Prⁱ) (1), where ISQ-Prⁱ = 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzosemiquinonate anion-radical, AP-Prⁱ = 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-amidophenolate dianion, has been prepared by the reaction of Mn₂(CO)₁₀ with free 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone (IBQ-Prⁱ) in the molar ratio 1:4 in toluene. In contrast to manganese, rhenium carbonyl reacts with o-iminobenzoquinone to form complex Re^II(ISQ-Prⁱ)₂(CO)₂ (2) with the retention of two carbonyls in coordination sphere of rhenium. The complexes have been characterized by IR, UV-Vis, and EPR spectroscopies. Molecular structures of compounds 1 and 2 have been determined by single-crystal X-ray crystallography. Compound 1 is centro-symmetric square-planar molecule with delocalized mixed valent state of AP-Prⁱ and ISQ-Prⁱ ligands. EPR spectrum of 1 in solid at 300-77 K is typical for manganese complexes with S = 3/2 state. The effective magnetic moment of 1 is 1.96 μ_B at temperature 5 K as it was established by variable-temperature magnetic susceptibility measurements. Six-coordinate octahedral complex 2 possesses an S = 1/2 ground state, which is attained via strong intramolecular antiferromagnetic interaction between t_2g orbital unpaired electron of the low spin Re^II ion and the unpaired electron on π-orbital of the radical ligand.     

Tetrameric structure of thermostable direct hemolysin from vibrio parahaemolyticus revealed by ultracentrifugation, small-angle X-ray scattering and electron microscopy

The thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus. We have characterized the conformational properties of TDH by small-angle X-ray scattering (SAXS), ultracentrifugation and transmission electron microscopy. Sedimentation equilibrium and velocity studies revealed that the protein is tetrameric in aqueous solvents. The Guinier plot derived from SAXS data provided a radius of gyration of 29.0 Å. The elongated pattern with a shoulder of a pair distance distribution function derived from SAXS data suggested the presence of molecules with an anisotropic shape having a maximum diameter of 98 Å. Electron microscopic image analysis of the negatively stained TDH oligomer showed the presence of C₄ symmetric particles with edge and diagonal lengths of 65 Å and 80 Å, respectively. Shape reconstruction was carried out by ab initio calculations using the SAXS data with a C₄ symmetric approximation. These results suggested that the tetrameric TDH assumes an oblate structure. The hydrodynamic parameters predicted from the ab initio model differed slightly from the experimental values, suggesting the presence of flexible segments.     

Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy

Noscapine biosynthesis in opium poppy is thought to occur via N-methylcanadine, which would be produced through 9-O-methylation of (S)-scoulerine, methylenedioxy bridge formation on (S)-tetrahydrocolumbamine, and N-methylation of (S)-canadine. Only scoulerine 9-O-methyltransferase has been functionally characterized. We report the isolation and characterization of a cytochrome P450 (CYP719A21) from opium poppy that converts (S)-tetrahydrocolumbamine to (S)-canadine. Recombinant CYP719A21 displayed strict substrate specificity and high affinity (K_m = 4.63 ± 0.71 μM) for (S)-tetrahydrocolumbamine. Virus-induced gene silencing of CYP719A21 caused a significant increase in (S)-tetrahydrocolumbamine accumulation and a corresponding decrease in the levels of putative downstream intermediates and noscapine in opium poppy plants.     

Quantification of cis and trans influences in [PtX(PPh3)3] complexes. A P NMR study

In [PtX(PPh₃)₃]⁺ complexes (X = F, Cl, Br, I, AcO, NO₃, NO₂, H, Me) the mutual cis and trans influences of the PPh₃ groups can be considered constants in the first place, therefore the one bond Pt-P coupling constants of P_(cis) and P_(trans) reflect the cis and trans influences of X. The compounds [PtBr(PPh₃)₃](BF₄) (2), [PtI(PPh₃)₃](BF₄) (3), [Pt(AcO)(PPh₃)₃](BF₄) (4), [Pt(NO₃)(PPh₃)₃](BF₄) (5), and the two isomers [Pt(NO₂-O)(PPh₃)₃](BF₄) (6a) and [Pt(NO₂-N)(PPh₃)₃](BF₄) (6b) have been newly synthesised and the crystal structures of 2 and 4·CH₂Cl₂·0.25C₃H₆O have been determined. From the ¹J_PtP values of all compounds we have deduced the series: I > Br > Cl > NO₃ > ONO > F > AcO > NO₂ > H > Me (cis influence) and Me > H > NO₂ > AcO > I > ONO > Br > Cl > F > NO₃ (trans influence). These sequences are like those obtained for the (neutral) cis- and trans-[PtClX(PPh₃)₂] derivatives, showing that there is no dependence on the charge of the complexes. On the contrary, the weights of both influences, relative to those of X = Cl, were found to depend on the charge and nature of the complex.     

Physiological,morphological and anatomical trait variations between winter and summer leaves of Cistus species

Morphological, anatomical and physiological summer and winter leaf traits of Cistus incanus subsp. incanus, C. salvifolius and C. monspeliensis growing at the Botanical garden of Rome were analyzed. With regard to differences between summer and winter leaves of the considered species, leaf thickness (L) was 21% higher in summer than in winter leaves (mean of the considered species) and this increase was mostly the result of the increased palisade parenchyma thickness over the spongy parenchyma one (24 and 16% higher in summer than in winter leaves, respectively). Leaf mass area (LMA) and leaf tissue density (LTD) were 38% and 17% higher in summer than in winter leaves, respectively (mean of the considered species). The photosynthetic rate (P_N), stomatal conductance (g_s) and chlorophyll content (Chl) of summer leaves were 54%, 17% and 14% lower, respectively, than in winter leaves. C. monspeliensis summer leaves had the highest LMA, LTD, adaxial cuticle thickness (14.6 ± 1.8 mg cm⁻², 1091 ± 94 mg cm⁻³, and 5.8 ± 1.7 μm, respectively) and the lowest mesophyll intercellular spaces (f_ias 38 ± 3%). Moreover, C. monspeliensis had the highest P_N in summer (2.6 ± 0.1 μmol m⁻² s⁻¹) and C. incanus the highest P_N and WUE (84% and 59% higher than the other species) in the favorable period, associated to a higher f_ias (42 ± 2%). C. salvifolius had the highest P_N (54% higher than the other species) in winter. The plasticity index could allow a better interpretation of the habitat preference of the considered species. The physiological plasticity (PI_p = 0.39, mean value of the considered species) was higher than the morphological (PI_m = 0.22, mean value) and anatomical (PI_a = 0.13, mean value) plasticity. Moreover, among the considered species, C. salvifolius and C. incanus are characterized by a larger PI_a (0.14, mean value) which seems to be correlated with their wider ecological distribution and the more favorable conditions of the environments where they naturally occur. The highest PI_m (0.29) of C. monspeliensis indicates that it can play a high adaptive role in highly stressed environments, like fire degraded Mediterranean areas in which it occurs.     

Interrelationships of Meloidogyne Species with Flue-cured Tobacco

Microplot and field experiments were conducted to determine relationships of population densities of Meloidogyne spp. to performance of flue-cured tobacco. A 3-yr microplot study of these interactions involved varying initial nematode numbers (P_i).and use of ethoprop to re-establish ranges of nematode densities. Field experiments included various nematicides at different locations. Regression analyses of microplot data from a loamy sand showed that cured-leaf yield losses on ''Coker 319'' for each 10-fold increase in P_i were as follows: M. javanica and M. arenaria—-13-19%; M. incognita—5-10%; M. hapla—3.4-5%; and 3% for M. incognita on resistant ''Speight G-28'' tobacco. A P_i of 750 eggs and larvae/500 cm³ of soil of all species except M. hapla caused a significant yield loss; only large numbers of M. hapla effected a loss. M. arenaria was the most tolerant species to ethoprop. Root-gall indices for microplot and most field-nematicide tests also were correlated negatively with yield. Relationships of P_i(s) and necrosis indices to yield were best characterized by linear regression models, whereas midseason numbers of eggs plus larvae (P_m) and sometimes gall indices vs. yield were better characterized by quadratic models. The relation of field P_m and yield was also adequately described by the Seinhorst model. Degrees of root galling, root necrosis, yield losses, and basic rates of reproduction on tobacco generally increased from M. hapla to M. incognita to M. arenaria to M. javanica.     

Community metabolism and buffering capacity of nitrogen in a ruppia cirrhosa meadow

Fluxes of oxygen, inorganic nitrogen (DIN) and denitrification (isotope pairing) were measured from January 1997 to February 1998 via intact cores incubation in a shallow brackish area within the eutrophic Valli di Comacchio (northern Adriatic coast, Italy). Rates were measured in the light and in the dark in sediments colonized by the rooted macrophyte Ruppia cirrhosa and in adjacent sediments with benthic microalgae. Ruppia biomass (25-414 g DW m^− 2) exhibited a seasonal evolution whilst that of microphytobenthos (12-66 mg chl a m^− 2) was more erratic. Net (NP) and gross (GP) primary productivity was 1.15 and 6.89 mol C m^− 2y^− 1 for bare and 25.4 and 51.7 mol C m^− 2y^− 1 for Ruppia vegetated sediments. Nitrogen pools in Ruppia standing stock varied from 43.6 to 631.4 (annual average 201.2) mmol N m^− 2; the macrophyte N content was correlated with DIN concentration in the water column. Estimated N pool in microphytobenthos was one order of magnitude lower (from 2.4 to 14.5 mmol N m^− 2, annual average 7.2). Theoretical DIN assimilation calculated from NP was 127.8 and 1112.6 mmol N m^− 2y^− 1 whilst that calculated from GP was 765 and 2282 mmol N m^− 2y^− 1 for microphytobenthos and Ruppia respectively. Measured annual fluxes of DIN were 974.6 and − 577 mmol N m^− 2y^− 1 in bare and Ruppia vegetated sediments meaning that the two sites were a source and sink for DIN and that from 25 to 50% of Ruppia annual DIN requirements came from the water column. During the period of this study total denitrification was lower in the macrophyte colonized (92.3 mmol N m^− 2y^− 1) compared to bare sediments (163.3 mmol N m^− 2y^− 1) as a probable consequence of higher competition between denitrifiers and phanerogams. At both sites the ratio between denitrification of water column nitrate (D_W) and denitrification coupled to nitrification (D_N) was >1.6 due to little oxygen penetration in reducing sediments (< 1.2 mm) and scarce nitrification activity. D_W (0-35 µmol N m^− 2h^− 1) was significantly correlated with water column NO₃⁻  (2-16 µM). Theoretical DIN assimilation to denitrification ratio varied from 12.0 to 24.8 for Ruppia vegetated and from 0.8 to 4.7 for unvegetated sediments.At Valle Smarlacca, Ruppia may influence nitrogen cycling by incorporating large DIN pools in biomass which is scattered in surrounding areas and fuels intense bacterial activity. With increasing anthropogenic nutrient input and insignificant organic matter export in the open sea the already severe eutrophic conditions are enhanced and may accelerate the decline of the macrophyte meadow.