Spectroscopic methods combined with density functional calculations are used to develop a detailed bonding description of the mu(4)-sulfide bridged tetranuclear Cu(Z) cluster in N(2)O reductase. The ground state of Cu(Z) has the 1Cu(II)/3Cu(I) configuration. The single electron hole dominantly resides on one Cu atom (Cu(I)) and partially delocalizes onto a second Cu atom (Cu(II)) via a Cu(I)-S-Cu(II) sigma/sigma superexchange pathway which is manifested by a Cu(II) --> Cu(I) intervalence transfer transition in absorption. The observed excited-state spectral features of Cu(Z) are dominated by the S --> Cu(I) charge-transfer transitions and Cu(I) based d-d transitions. The intensity pattern of individual S --> Cu(I) charge-transfer transitions reflects different bonding interactions of the sulfur valence orbitals with the four Cu's in the Cu(Z) cluster, which are consistent with the individual Cu-S force constants obtained from a normal coordinate analysis of the Cu(Z) resonance Raman frequencies and profiles. The Cu(I) d orbital splitting pattern correlates with its distorted T-shaped ligand field geometry and accounts for the observed low g( parallel ) value of Cu(Z) in EPR. The dominantly localized electronic structure description of the Cu(Z) site results from interactions of Cu(II) with the two additional Cu's of the cluster (Cu(III)/Cu(IV)), where the Cu-Cu electrostatic interactions lead to hole localization with no metal-metal bonding. The substrate binding edge of Cu(Z) has a dominantly oxidized Cu(I) and a dominantly reduced Cu(IV). The electronic structure description of Cu(Z) provides a strategy to overcome the reaction barrier of N(2)O reduction at this Cu(I)/Cu(IV) edge by simultaneous two-electron transfer to N(2)O in a bridged binding mode. One electron can be donated directly from Cu(IV) and the other from Cu(II) through the Cu(II)-S-Cu(I) sigma/sigma superexchange pathway. A frontier orbital scheme provides molecular insight into the catalytic mechanism of N(2)O reduction by the Cu(Z) cluster.

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.

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Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = (1)/(2)) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O(2)(-) or OH(-)) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW (17)O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H(2)(17)O, on the active Ni-C (S = (1)/(2)) form prepared by H(2)-reduction of Ni-A in H(2)(17)O, and also on Ni-A formed by reoxidation of Ni-C in H(2)(17)O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H(2)(17)O solvent. However, after a reduction/reoxidation cycle (Ni-A --> Ni-C --> Ni-A), an (17)O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A((17)O) shows that the incorporated (17)O has a roughly axial hyperfine tensor, A((17)O) approximately [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a(iso)((17)O) approximately 11 MHz) of the hyperfine interaction indicates that a solvent-derived (17)O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O(2)(-) or OH(-)) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small (57)Fe hyperfine coupling seen previously for Ni-A (A((57)Fe) approximately 0.9 MHz) is now shown to persist in Ni-C, A((57)Fe) approximately 0.8 MHz. However, the (17)O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme's activity.

Spectroscopy coupled with density functional calculations has been used to define the spin state, oxidation states, spin distribution, and ground state wave function of the mu4-sulfide bridged tetranuclear CuZ cluster of nitrous oxide reductase. Initial insight into the electronic contribution to N2O reduction is developed, which involves a sigma superexchange pathway through the bridging sulfide.

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This article aims to study hydrogen production/consumption in Desulfovibrio (D.) desulfuricans strain New Jersey, a sulfate reducer isolated from a medium undergoing active biocorrosion and to compare its hydrogen metabolism with two other Desulfovibrio species, D. gigas and D. vulgaris Hildenborough. Hydrogen production was followed during the growth of these three bacterial species under different growth conditions: no limitation of sulfate and lactate, sulfate limitation, lactate limitation, pyruvate/sulfate medium and in the presence of molybdate. Hydrogen production/consumption by D. desulfuricans shows a behavior similar to that of D. gigas but a different one from that of D. vulgaris, which produces higher quantities of hydrogen on lactate/sulfate medium. The three species are able to increase the hydrogen production when the sulfate became limiting. Moreover, in a pyruvate/sulfate medium hydrogen production was lower than on lactate/sulfate medium. Hydrogen production by D. desulfuricans in presence of molybdate is extremely high. Hydrogenases are key enzymes on production/consumption of hydrogen in sulfate reducing organisms. The specific activity, number and cellular localization of hydrogenases vary within the three Desulfovibrio species used in this work, which could explain the differences observed on hydrogen utilization.

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The unambiguous assignment of the nuclear magnetic resonance (NMR) signals of the α-substituents of the haems in the tetrahaem cytochrome isolated from Shewanella frigidimarina NCIMB400, was made using a combination of homonuclear and heteronuclear experiments. The paramagnetic 13C shifts of the nuclei directly bound to the porphyrin of each haem group were analysed in the framework of a model for the haem electronic structure. The analysis yields g-tensors for each haem, which allowed the assignment of some electron paramagnetic resonance (EPR) signals to specific haems, and the orientation of the magnetic axes relative to each haem to be established. The orientation of the axial ligands of the haems was determined semi-empirically from the NMR data, and the structural results were compared with those of the homologous tetrahaem cytochrome from Shewanella oneidensis MR-1 showing significant similarities between the two proteins.

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