Multiple Roles of Component Proteins in Bacterial Multicomponent Monooxygenases: Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas sp. OX1.

Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In this study, we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H(2)O(2)) generation. Single-turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was ∼50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single-turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to H(2)O(2) formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed

Radiosensitization of DNA in presence of Pt(II)-based compounds

X-ray irradiation of plasmid DNA in presence of platinum (II)-based compounds was carried out in order to assess the radiosensitization capabilities of these drugs. In present investigations pBR322 plasmid DNA was used to monitor effectiveness of chosen compounds in inducing strand breaks. Samples were incubated in presence of potential radiosensitisers: platinum (II) bromide and cis-diamminedibromoplatinum (II). The results were examined against a common cancer chemotherapy drug cis-diamminedichloroplatinum (II). It was found that platinum (II) bromide can greatly increase the levels of single- and double-strand break formation observed in the irradiated samples with respect to the samples containing platinum as a radiosensitizer only, possessing very little chemotherapeutic activity. The suggested drugs exhibit much higher level of radiosensitivity than widely used cisplatin and thus may be good candidates for cancer treatment

Short term development of intracontinental rifts, with reference to the late Quaternary of the Rukwa Rift (East African Rift System)

Relatively low-resolution seismic data and high contemporaneous rift topography normally limit quantitative analysis of normal faults in rifts. The availability of a recently collected high-resolution reflection seismic survey in the SE part of the presently active Rukwa Rift (East African Rift System) coupled with high sedimentation rates in the submerged part of the rift makes detailed quantitative analysis possible. High-resolution (down to about 1 m) seismic data penetrate ca. 300 m (representing about 150,000 years) of the uppermost sediments and show fine details of normal faulting and related structures. Displacements on the faults, which occur in the hanging wall of the major rift boundary fault, range from a few metres up to 100 m. They show increasing displacements with depth and characteristic rollover folding of the hanging walls. Fault propagation occurred upwards through the rapidly accumulating lake sediments and was preceded by flexuring and folding of the sediments. The geometry of the seismic sequences with alternating wedge-shaped and tabular units reflects pulsed activity on the faults. Fault displacement rates of up to 1.6 mm yr-1 have been estimated. Each extensional pulse has a duration of 1000s to 10,000s of years. The quiescent stages have a similar duration. Up to 2.5% extension has occurred during the recent evolution

Multiple Roles of Component Proteins in Bacterial Multicomponent Monooxygenases: Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas sp. OX1.

Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In this study, we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H(2)O(2)) generation. Single-turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was ∼50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single-turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to H(2)O(2) formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed

Preparation and X-ray structures of metal-free, dicobalt and dimanganese forms of soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath)

A three-component soluble methane monooxygenase (sMMO) enzyme system catalyzes the hydroxylation of methane to methanol at a carboxylate-bridged diiron center housed in the a-subunit of the hydroxylase (MMOH). Catalysis is facilitated by the presence of a regulatory protein (MMOB) and inhibited by MMOD, a protein of unknown function encoded in the sMMO operon. Both MMOB and MMOD are presumed to bind to the same region of the MMOH a-subunit. A colorimetric method for monitoring removal of Fe(II) from MMOH was developed using 1,10-phenanthroline and yields apo MMOH wit

Determination by X-ray absorption spectroscopy of the Fe-Fe separation in the oxidized form of the hydrxylase of methane monooxygenase alone and in the presence of MMOD

The diiron active site in the hydroxylase of Methylococcus capsulatus (Bath) methane monooxygenase (MMOH) has been studied in the oxidized form by X-ray absorption spectroscopy (XAS). Previous investigations by XAS and X-ray crystallography have identified two different distances (3.0 and 3.4 Å) between the two Fe atoms in the dinuclear site. The present study has employed a systematic extended X-ray absorption fine structure (EXAFS) fitting methodology, utilizing known and simulated active site and relevant model structures, to determine unambiguously the Fe-Fe separation in the oxidized form of MMOH. Consistent and unique fits were only possible for an Fe-Fe distance of 3.0 Å. This methodology was then applied to study potential changes in the active site local structure in the presence of MMOD, a protein of unknown function in multicomponent MMO. Fe K-edge and EXAFS analyses revealed negligible changes in the diiron site electronic and geometric structure upon addition of MMOD to oxidized MMOH

Structural restoration of Cretaceous-Cenozoic (post-rift) palaeobathymetry in the northern North Sea

Palaeobathymetric variations and palaeobasin shape are important for recognition of tectonic phases and distribution of sediment. We present an approach for restoring palaeobathymetry from interpreted, depth-converted seismic sections. The method involves section balancing / restoration techniques for extensional regimes and seismic sequence stratigraphy. The method is based on regional profiles that cover the entire basin using fixed water-depth points such as coal layers, shallow marine sand and subaerial unconformities, which are used to calibrate areas along the profile. Geometric information in the under- and overlying seismic sequences is used to shape the palaeobasin. The palaeowater-depths and basin gradients of the Cretaceous and Tertiary post-rift interval in the northern North Sea have been restored. The results show a highly segmented basin with high gradients and locally large water-depths in the earliest Cretaceous. A shallowing and a lowering of the basin gradients occurred in the early Cretaceous. A deepening and widening of the basin was initiated in the middle Cretaceous. During the late Cretaceous the water-depth and the basin gradients decreased. A deepening occurred in the Palaeogene resulting in high gradients on the flanks of the basin. The basin became shallower and the relief became smaller in the early Neogene. The basin deepened in the late Neogene and then became shallower to the present day. The palaeobathymetric trends correlate well with results obtained from micropalaeontological analysis of palaeowater-depth in the same area