New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway,
Folgosa, F., Cordas C. M., Santos J. A., Pereira A. S., Moura J. J., Tavares P., and Moura I.
, Biochem J, Sep 15, Volume 438, Number 3, p.485-94, (2011)
AbstractSORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-centre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 x 10 M(1) . s(1) and 1.3 x 10 M(1) . s(1) for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2 x 10 M(1) . s(1) for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.
Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria,
Mukhopadhyay, A., Kladova A. V., Bursakov S. A., Gavel O. Y., Calvete J. J., Shnyrov V. L., Moura I., Moura J. J., Romao M. J., and Trincao J.
, J Biol Inorg Chem, Jan, Volume 16, Number 1, p.51-61, (2011)
AbstractAdenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn(2+), Zn-AK; Co(2+), Co-AK; and Fe(2+), Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 A, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.
Artefacts induced on c-type haem proteins by electrode surfaces,
Paes de Sousa, P. M., Pauleta S. R., Simoes Goncalves M. L., Pettigrew G. W., Moura I., Moura J. J., and Correia dos Santos M. M.
, J Biol Inorg Chem, Feb, Volume 16, Number 2, p.209-15, (2011)
AbstractIn this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met-His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c(3) from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.
Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain,
Paes de Sousa, P. M., Rodrigues D., Timoteo C. G., Simoes Goncalves M. L., Pettigrew G. W., Moura I., Moura J. J., and Correia dos Santos M. M.
, J Biol Inorg Chem, Aug, Volume 16, Number 6, p.881-8, (2011)
AbstractThe activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 +/- 1) x 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.
Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis,
Grazina, R., de Sousa P. M., Brondino C. D., Carepo M. S., Moura I., and Moura J. J.
, Bioelectrochemistry, Aug, Volume 82, Number 1, p.22-8, (2011)
AbstractThe redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe-4S] and one [4Fe-4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S](+1/0) (E(0')=-158+/-5mV); [4Fe-4S](+2/+1) (E(0')=-474+/-5mV) and [3Fe-4S](0/-2) (E(0')=-660+/-5mV). The effect of pH on these signals showed that the reduced [3Fe-4S](0) cluster has a pK'(red)(')=5.1+/-0.1, the [4Fe-4S](+2/+1) centre is pH independent, and the [3Fe-4S](0/-2) reduction is accompanied by the binding of two protons. The ability of the [3Fe-4S](0) cluster to be converted into a new [4Fe-4S] cluster was proven. The redox potential of the original [4Fe-4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70mV) of the redox potential of the original centre. Being most [Fe-S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed.
Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to Improve the selectivity of nitrite biosensing,
A.S., Serra, S. Jorge, C. Silveira, J.J.G. Moura, E. Jubete, E. Ochoteco, and G. Almeida M.
, Anal Chim Acta, Volume 693, p.41-46, (2011)
Nitrite biosensing using cytochrome c nitrite reductase: Towards a disposable strip electrode,
Correia, C., Rodrigues M., Silveira C. M., Moura J. J. G., Ochoteco E., Jubete E., and Almeida M. G.
, Biomedical Engineering Systems and Technologies. Series: Communications in Computer and Information Science, (2011)
Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing,
Serra, A. S., Jorge S. R., Silveira C. M., Moura J. J. G., Jubete E., Ochoteco E., Cabañero G., Grande H., and Almeida M. G.
, Analytica Chimica Acta, Volume 693, Number 1–2, p.41-46, (2011)
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