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Rearrangement of Mo-Cu-S Cluster Reflects the Structural Instability of Orange Protein Cofactor, Maiti, B. K., Avilés T., Carepo M. S., Moura I., S.R. Pauleta, and Moura J. J. G. , Z Anorg Allg Chem, Volume 639, p.1361-1364, (2013)
Recent advances into vanadyl, vanadate and decavanadate interactions with actin, Ramos, S., Moura J. J. G., and Aureliano M. , Metallomics, Volume 4, Issue 1, Number 1, p.16-22, (2012)
REDOX AND SPIN-STATE CONTROL OF THE ACTIVITY OF A DIHEME CYTOCHROME-C PEROXIDASE - SPECTROSCOPIC STUDIES, Prazeres, S., Moura I., Gilmour R., Pettigrew G., Ravi N., and Huynh B. H. , Nuclear Magnetic Resonance of Paramagnetic Macromolecules, Volume 457, p.141-163, (1995) Abstract
Redox chemistry of low-pH forms of tetrahemic cytochrome c3, Santos, M., Dos Santos M. M., Goncalves M. L., Costa C., Romao J. C., and Moura J. J. , J Inorg Biochem, Dec, Volume 100, Number 12, p.2009-16, (2006) AbstractWebsite

Desulfovibrio vulgaris Hildenborough cytochrome c(3) contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c(3) can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV-visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.

Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. Mossbauer and EPR characterization of the metal centers, Teixeira, M., Moura I., Xavier A. V., Moura J. J., Legall J., Dervartanian D. V., Peck, H. D. Jr., and Huynh B. H. , J Biol Chem, Oct 5, Volume 264, Number 28, p.16435-50, (1989) AbstractWebsite

The hydrogenase (EC of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and Mossbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.

Redox potential measurements of the Mycobacterium tuberculosis heme protein KatG and the isoniazid-resistant enzyme KatG(S315T): insights into isoniazid activation, Wengenack, N. L., Lopes H., Kennedy M. J., Tavares P., Pereira A. S., Moura I., Moura J. J., and Rusnak F. , Biochemistry, Sep 19, Volume 39, Number 37, p.11508-13, (2000) AbstractWebsite

Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in >50% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe(3+)/Fe(2+) couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and II or oxyferrous KatG.

Redox properties and activity studies on a nickel-containing hydrogenase isolated from a halophilic sulfate reducer Desulfovibrio salexigens, Teixeira, M., Moura I., Fauque G., Czechowski M., Berlier Y., Lespinat P. A., Legall J., Xavier A. V., and Moura J. J. , Biochimie, Jan, Volume 68, Number 1, p.75-84, (1986) AbstractWebsite

A soluble hydrogenase from the halophilic sulfate reducing bacterium Desulfovibrio salexigens, strain British Guiana (NCIB 8403) has been purified to apparent homogeneity with a final specific activity of 760 mumoles H2 evolved/min/mg (an overall 180-fold purification with 20% recovery yield). The enzyme is composed of two non-identical subunits of molecular masses 62 and 36 kDa, respectively, and contains approximately 1 Ni, 12-15 Fe and 1 Se atoms/mole. The hydrogenase shows a visible absorption spectrum typical of an iron-sulfur containing protein (A400/A280 = 0.275) and a molar absorbance of 54 mM-1cm-1 at 400 nm. In the native state (as isolated, under aerobic conditions), the enzyme is almost EPR silent at 100 K and below. However, upon reduction under H2 atmosphere a rhombic EPR signal develops at g-values 2.22, 2.16 and around 2.0, which is optimally detected at 40 K. This EPR signal is reminiscent of the nickel signal C (g-values 2.19, 2.16 and 2.02) observed in intermediate redox states of the well characterized D. gigas nickel containing hydrogenase and assigned to nickel by 61 Ni isotopic substitution (J.J.G. Moura, M. Teixeira, I. Moura, A.V. Xavier and J. Le Gall (1984), J. Mol. Cat., 23, 305-314). Upon longer incubation with H2 the "2.22" EPR signal decreases. During the course of a redox titration under H2, this EPR signal attains a maximal intensity around--380 mV. At redox states where this "2.22" signal develops (or at lower redox potentials), low temperature studies (below 10 K) reveals the presence of other EPR species with g-values at 2.23, 2.21, 2.14 with broad components at higher fields. This new signal (fast relaxing) exhibits a different microwave power dependence from that of the "2.22" signal, which readily saturates with microwave power (slow relaxing). Also at low temperature (8 K) typical reduced iron-sulfur EPR signals are concomitantly observed with gmed approximately 1.94. The catalytic properties of the enzyme were also followed by substrate isotopic exchange D2/H+ and H2 production measurements.

Redox properties of cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774, Costa, C., Moura J. J., Moura I., Wang Y., and Huynh B. H. , J Biol Chem, Sep 20, Volume 271, Number 38, p.23191-6, (1996) AbstractWebsite

The dissimilatory nitrite reductase from Desulfovibrio desulfuricans ATCC 27774 catalyzes the reduction of nitrite to ammonia. Previous spectroscopic investigation revealed that it is a hexaheme cytochrome containing one high spin ferric heme and five low spin ferric hemes in the oxidized enzyme. The current study uses the high resolution of Mossbauer spectroscopy to obtain redox properties of the six heme groups. Correlating the Mossbauer findings with the EPR data reveals the pairwise spin-spin coupling among four of the heme groups. The other two hemes are found to be magnetically isolated. Reduction with dithionite and reaction with CO further indicate that only the high spin heme is capable of binding small exogenous ligands. These results confirm our previous finding that Desulfovibrio desulfuricans nitrite reductase contains six heme groups and that the high spin ferric heme is the substrate and inhibitor binding site.

Redox properties of Desulfovibrio gigas [Fe3S4] and [Fe4S4] ferredoxins and heterometal cubane-type clusters formed within the [Fe3S4] core. Square wave voltammetric studies, Moreno, C., Macedo A. L., Moura I., Legall J., and Moura J. J. , J Inorg Biochem, Feb 15, Volume 53, Number 3, p.219-34, (1994) AbstractWebsite

The same polypeptide chain (58 amino acids, 6 cysteines) is used to build up two ferredoxins in Desulfovibrio gigas a sulfate reducing organism. Ferredoxin II (FdII) contains a single [Fe3S4] core and ferredoxin I (FdI) mainly a [Fe4S4] core. The [Fe3S4] core can readily be interconverted into a [Fe4S4] complex (J.J.G. Moura, I. Moura, T.A. Kent, J.D. Lipscomb, B.H. Huynh, J. LeGall, A.V. Xavier, and E. Munck, J. Biol. Chem. 257, 6259 (1982)). This interconversion process suggested that the [Fe3S4] core could be used as a synthetic precursor for the formation of heterometal clusters. Co, Zn, Cd, and Ni derivatives were produced (I. Moura, J.J.G. Moura, E. Munck, V. Papaephthymiou, and J. LeGall, J. Am. Chem. Soc. 108, 349 (1986), K. Sureurs, E. Munck, I. Moura, J.J.G. Moura, and J. LeGall, J. Am. Chem. Soc. 109, 3805 (1986), and A.L. Macedo, I. Moura, J.J.G. Moura, K. Surerus, and E. Munck, unpublished results). The redox properties of a series of heterometal clusters (MFe3S4] are assessed using direct electrochemistry (square wave voltammetry--SWV) promoted by Mg(II) at a glassy carbon electrode (derivatives: Cd (-495 mV), Fe (-420 mV), Ni (-360 mV), and Co (-245 mV) vs normal hydrogen electrode (NHE)). In parallel, the electrochemical behavior (cyclic voltammetry--CV, differential pulse voltammetry--DPV and SWV) of FdI and FdII were investigated as well as the cluster interconversion process. In addition to the +1/0 (3Fe cluster) and +2/+1 (4Fe cluster) redox transitions, a very negative redox step, at -690 mV, was detected for the 3Fe core, reminiscent of a postulated further 2e- reduction step, as proposed for D. africanus ferredoxin III by F.A. Armstrong, S.J. George, R. Cammack, E.C. Hatchikian, and A.J. Thomson, Biochem. J. 264, 265 (1989). The electrochemical redox potential values are compared with those determined by independent methods (namely by electron paramagnetic resonance (EPR) and visible spectroscopy).

Redox properties of the diheme cytochrome c4 from Azotobacter vinelandii and characterisation of the two hemes by NMR, MCD and EPR spectroscopy, Gadsby, P. M., Hartshorn R. T., Moura J. J., Sinclair-Day J. D., Sykes A. G., and Thomson A. J. , Biochim Biophys Acta, Jan 19, Volume 994, Number 1, p.37-46, (1989) AbstractWebsite

From biphasic stopped-flow kinetic studies it has been established that the two heme centres of cytochrome c4 from Azotobacter vinelandii undergo redox change with [Co(terpy)2]3+/2+ (260 mV) at different rates. Rate constants for oxidation and reduction at pH 7.5 give reduction potentials for the two heme centres in agreement with previous values from spectrophotometric titrations (263 and 317 mV). From NMR studies on the fully reduced protein two sharp methyl methionine resonances are observed at -3.16 and -3.60 ppm, consistent with axial methionine coordination. On titration with [Fe(CN)6]3- the -3.16 ppm resonance is the first to disappear, and is assigned to the less positive reduction potential. Line-broadening effects are observed on partial oxidation, which are dominated by intermolecular processes in an intermediate time-range exchange process. The hemes of the oxidised protein are distinguishable by EPR g-values of 3.64 and 3.22. The former is of interest because it is at an unusually low field for histidine/methionine coordination, and has an asymmetric or ramp shape. The latter assigned to the low potential heme is similar to that of a cytochrome c551. The MCD spectra of the fully oxidised protein are typical of low-spin Fe(III) heme centres, with a negative peak at 710 nm characteristic of methionine coordination, and an NIR peak at 1900 nm characteristic of histidine/methionine (axial) coordination. Of the four histidines per molecule only two undergo diethyl pyrocarbonate (DEPC) modification.

Redox states of cytochrome c3 in the absence and presence of ferredoxin, Moura, J. J., Xavier A. V., Cookson D. J., Moore G. R., and Williams R. J. , FEBS Lett, Sep 15, Volume 81, Number 2, p.275-80, (1977) AbstractWebsite
Redox studies on rubredoxins from sulphate and sulphur reducing bacteria, Moura, I., Moura J. J., Santos M. H., Xavier A. V., and Legall J. , FEBS Lett, Nov 15, Volume 107, Number 2, p.419-21, (1979) AbstractWebsite
Redox thermodynamics of low-potential iron-sulfur proteins, Battistuzzi, G., D'Onofrio M., Borsari M., Sola M., Macedo A. L., Moura J. J., and Rodrigues P. , J Biol Inorg Chem, Dec, Volume 5, Number 6, p.748-60, (2000) AbstractWebsite

The enthalpy and entropy changes associated with protein reduction (deltaHdegrees,(rc), deltaSdegrees,(rc)) were determined for a number of low-potential iron-sulfur proteins through variable temperature direct electrochemical experiments. These data add to previous estimates making available, overall, the reduction thermodynamics for twenty species from various sources containing all the different types of metal centers. These parameters are discussed with reference to structural data and calculated electrostatic metal-environment interaction energies, and redox properties of model complexes. This work, which is the first systematic investigation on the reduction thermodynamics of Fe-S proteins, contributes to the comprehension of the determinants of the differences in reduction potential among different protein families within a novel perspective. Moreover, comparison with analogous data obtained previously for electron transport (ET) metalloproteins with positive reduction potentials, i.e., cytochromes c, blue copper proteins, and HiPIPs, helps our understanding of the factors controlling the reduction potential in ET species containing different metal cofactors. The main results of this work can be summarized as follows.

Reduction of ascorbate free radical by the plasma membrane of synaptic terminals from rat brain, Samhan-Arias, A. K., Duarte R. O., Martin-Romero F. J., Moura J. J., and Gutierrez-Merino C. , Arch Biochem Biophys, Jan 15, Volume 469, Number 2, p.243-54, (2008) AbstractWebsite

Synaptic plasma membranes (SPMV) decrease the steady state ascorbate free radical (AFR) concentration of 1mM ascorbate in phosphate/EDTA buffer (pH 7), due to AFR recycling by redox coupling between ascorbate and the ubiquinone content of these membranes. In the presence of NADH, but not NADPH, SPMV catalyse a rapid recycling of AFR which further lower the AFR concentration below 0.05 microM. These results correlate with the nearly 10-fold higher NADH oxidase over NADPH oxidase activity of SPMV. SPMV has NADH-dependent coenzyme Q reductase activity. In the presence of ascorbate the stimulation of the NADH oxidase activity of SPMV by coenzyme Q(1) and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q(1) and ascorbate/cytochrome c. The NADH:AFR reductase activity makes a major contribution to the NADH oxidase activity of SPMV and decreases the steady-state AFR concentration well below the micromolar concentration range.

Reduction of carbon dioxide by a molybdenum-containing formate dehydrogenase: a kinetic and mechanistic study, Maia, L. B., Fonseca L., Moura I., and Moura J. J. G. , J Am Chem Soc, Volume 138, p.8834-8846, (2016) Website
Reductive activation of aerobically purified Desulfovibrio vulgaris hydrogenase: Mossbauer characterization of the catalytic H cluster, Huynh, B. H., Tavares P., Pereira A. S., Moura I., and Moura J. J. G. , Biochemistry and Physiology of Anaerobic Bacteria, 2003, p.35-45, (2003) AbstractWebsite
Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli. A mass spectrometric study, Costa, C., Macedo A., Moura I., Moura J. J., Legall J., Berlier Y., Liu M. Y., and Payne W. J. , FEBS Lett, Dec 10, Volume 276, Number 1-2, p.67-70, (1990) AbstractWebsite

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.

Relations between mercury, methyl-mercury and selenium in tissues of Octopus vulgaris from the Portuguese Coast, Raimundo, Joana, Vale Carlos, Canario Joao, Branco Vasco, and Moura Isabel , Environmental Pollution, Jun, Volume 158, Number 6, p.2094-2100, (2010) AbstractWebsite

Mercury, methyl-mercury (MeHg) and selenium were determined in digestive gland and mantle of Octopus vulgaris, from three areas of the Portuguese coast. To our knowledge these are the first data on MeHg in cephalopods. Concentrations were higher in the digestive gland and percentage of MeHg in mantle. Enhanced Hg and MeHg levels were obtained in digestive gland of specimens from Olhao (3.1-7.4 and 2.0-5.0 mu g g(-1) respectively). Differences between areas may be partially related to Hg availability. Relationships between concentrations in mantle and digestive gland pointed to proportional increases of Hg and MeHg in tissues of specimens from Matosinhos and Cascais, but relatively constant values in mantle of individuals from Olhao (higher contamination). Se:Hg molar ratio in digestive gland was 32 and 30 in octopus from Matosinhos and Cascais, respectively, and 5.4 from Olhao. The proximity to the unit suggests demethylation as response to elevated MeHg levels in digestive gland. (C) 2010 Elsevier Ltd. All rights reserved.

Replacement of Methionine as the Axial Ligand of Achromobacter cycloclastes Cytochrome C554 at High pH Values Revealed by Absorption, EPR and MCD Spectroscopy, Saraiva, L. M., Thomson A. J., Lebrun N. E., Liu M. Y., Payne W. J., Legall J., and Moura I. , Biochemical and Biophysical Research Communications, Volume 204, Number 1, p.120-128, (1994) AbstractWebsite
Resonance Raman spectra of rubredoxin, desulforedoxin, and the synthetic analog Fe(S2-o-xyl)2: conformational effects, Yachandra, Vittal K., Hare Jeffrey, Moura I., and Spiro Thomas G. , Journal of the American Chemical Society, 1983/10/01, Volume 105, Number 21, p.6455-6462, (1983) AbstractWebsite
Resonance Raman spectra of rubredoxin: new assignments and vibrational coupling mechanism from iron-54/iron-56 isotope shifts and variable-wavelength excitation, Czernuszewicz, Roman S., Legall Jean, Moura Isabel, and Spiro Thomas G. , Inorganic Chemistry, 1986/02/01, Volume 25, Number 5, p.696-700, (1986) AbstractWebsite
Resonance Raman spectra of three-iron centers in ferredoxins from Desulfovibrio gigas, Johnson, M. K., Hare J. W., Spiro T. G., Moura J. J., Xavier A. V., and Legall J. , J Biol Chem, Oct 10, Volume 256, Number 19, p.9806-8, (1981) AbstractWebsite

The resonance Raman spectra of ferredoxins (Fd) I and II from Desulfovibrio gigas are reported using 4579 A Ar+ laser excitation. The (3Fe-3S) center in Fd II has a characteristic resonance Raman spectrum, readily distinguishable from those of (2Fe-2S) or (4Fe-4S) clusters. Reduction of Fd II produces a marked alteration in the resonance Raman spectrum. Fd I is shown to contain both (3Fe-3S) and (4Fe-4S) Fd-type clusters. The results illustrate the potential of resonance Raman spectroscopy in Fe-S cluster identification, even in cases where more than one cluster type is present.

Resonance Raman studies of nickel tetrathiolates and nickel-substituted rubredoxins and desulforedoxin, Huang, Yun Hua, Moura Isabel, Moura Jose J. G., Legall Jean, Park Jae Bum, Adams Michael W. W., and Johnson Michael K. , Inorganic Chemistry, 1993/02/01, Volume 32, Number 4, p.406-412, (1993) AbstractWebsite
Resonance Raman study of sirohydrochlorin and siroheme in sulfite reductases from sulfate reducing bacteria, Underwood-Lemons, Theresa, Moura Isabel, and To Yue Kwok , Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1157, Number 2, p.275-284, (1993) AbstractWebsite
Resonance Raman study on the iron-sulfur centers of Desulfovibrio gigas aldehyde oxidoreductase, Zhelyaskov, V., Yue K. T., Legall J., Barata B. A., and Moura J. J. , Biochim Biophys Acta, Oct 25, Volume 1252, Number 2, p.300-4, (1995) AbstractWebsite

Resonance Raman spectra of the molybdenum containing aldehyde oxidoreductase from Desulfovibrio gigas were recorded at liquid nitrogen temperature with various excitation wavelengths. The spectra indicate that all the iron atoms are organised in [2Fe-2S] type centers consistent with cysteine ligations. No vibrational modes involving molybdenum could be clearly identified. The features between 280 and 420 cm-1 are similar but different from those of typical plant ferredoxin-like [2Fe-2S] cluster. The data are consistent with the presence of a plant ferredoxin-like cluster (center I) and a unique [2Fe-2S] cluster (center II), as suggested by other spectroscopic studies. The Raman features of center II are different from those of other [2Fe-2S] clusters in proteins. In addition, a strong peak at ca. 683 cm-1, which is not present in other [2Fe-2S] clusters in proteins, was observed with purple excitation (406.7-413.1 nm). The peak is assigned to enhanced cysteinyl C-S stretching in center II, suggesting a novel geometry for this center.