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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) AbstractWebsite

SORs (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.

The primary structure of the beta subunit of Desulfovibrio desulfuricans (ATCC 27774) NiFe hydrogenase, Franco, R., Calvete J. J., Thole H. H., Raida M., Moura I., and Moura J. J. G. , Protein and Peptide Letters, Apr, Volume 4, Number 2, p.131-138, (1997) AbstractWebsite

The periplasmic [NiFe] hydrogenase isolated from Desulfovibrio (D.) desulfuricans (ATCC 27774) is a heterodimer of a 28 kDa (beta) and a 60 kDa (alpha) subunit. Here we report the complete amino acid sequence of the small (beta) polypeptide chain determined by Edman degradation of proteolytic fragments. Electrospray-ionization mass spectrometry of the native protein confirmed the sequencing results. The sequence is compared with that of D. gigas [NiFe] hydrogenase whose three-dimensional structure has been recently published.

Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mossbauer methods, Franco, R., Moura J. J., Moura I., Lloyd S. G., Huynh B. H., Forbes W. S., and Ferreira G. C. , J Biol Chem, Nov 3, Volume 270, Number 44, p.26352-7, (1995) AbstractWebsite

All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mossbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mossbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mossbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet. Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are delta = 1.36 +/- 0.03 mm/s and delta EQ = 3.04 +/- 0.06 mm/s, distinct from the larger delta EQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants, Franco, R., Pereira A. S., Tavares P., Mangravita A., Barber M. J., Moura I., and Ferreira G. C. , Biochemical Journal, May 15, Volume 356, p.217-222, (2001) AbstractWebsite

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

Characterization of D. desulfuricans (ATCC 27774) [NiFe] hydrogenase EPR and redox properties of the native and the dihydrogen reacted states, Franco, R., Moura I., Legall J., Peck, H. D. Jr., Huynh B. H., and Moura J. J. , Biochim Biophys Acta, Oct 4, Volume 1144, Number 3, p.302-8, (1993) AbstractWebsite

Redox intermediates of D. desulfuricans ATCC 27774 [NiFe] hydrogenase were generated under dihydrogen. Detailed redox titrations, coupled to EPR measurements, give access to the mid-point redox potentials of the iron-sulfur centers and of the Nickel-B signal that represents the ready form of the enzyme. The interaction between the dihydrogen molecule and the nickel centre was probed by the observation of an isotopic effect on the EPR signals detected in turnover conditions, by comparison of the H2O/H2 and D2O/D2-reacted samples.

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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.

Influence of the protein staining in the fast ultrasonic sample treatment for protein identification through peptide mass fingerprint and matrix-assisted laser desorption ionization time of flight mass spectrometry, Galesio, M., Vieira D. V., Rial-Otero R., Lodeiro C., Moura I., and Capelo J. L. , Journal of Proteome Research, May, Volume 7, Number 5, p.2097-2106, (2008) AbstractWebsite

The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra.

Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774, Gavel, O. Y., Kladova A. V., Bursakov S. A., Dias J. M., Texeira S., Shnyrov V. L., Moura J. J., Moura I., Romao M. J., and Trincao J. , Acta Crystallogr Sect F Struct Biol Cryst Commun, Jul 1, Volume 64, Number Pt 7, p.593-5, (2008) AbstractWebsite

Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC 2.7.7.4; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 A resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.

A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774, Gavel, O. Y., Bursakov S. A., Di Rocco G., Trincao J., Pickering I. J., George G. N., Calvete J. J., Shnyrov V. L., Brondino C. D., Pereira A. S., Lampreia J., Tavares P., Moura J. J., and Moura I. , J Inorg Biochem, May-Jun, Volume 102, Number 5-6, p.1380-95, (2008) AbstractWebsite

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas, Gavel, O. Y., Bursakov S. A., Pina D. G., Zhadan G. G., Moura J. J., Moura I., and Shnyrov V. L. , Biophys Chem, Jul 1, Volume 110, Number 1-2, p.83-92, (2004) AbstractWebsite

A novel adenylate kinase (AK) has recently been purified from Desulfovibrio gigas and characterized as a Co(2+)/Zn(2+)-containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these enzymes are normally devoid of metals. Here, we studied the conformational stability of holo- and apo-AK as a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy. The thermal unfolding of AK is a cooperative two-state process, and is sufficiently reversible in the 9-11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic model. The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the highly sensitive but integral DSC-method.

ATP sulfurylases from sulfate-reducing bacteria of the genus Desulfovibrio. A novel metalloprotein containing cobalt and zinc, Gavel, O. Y., Bursakov S. A., Calvete J. J., George G. N., Moura J. J., and Moura I. , Biochemistry, Nov 17, Volume 37, Number 46, p.16225-32, (1998) AbstractWebsite

Adenosine triphosphate sulfurylase catalyzes the formation of adenosine 5'-phosphosulfate from adenosine triphosphate and sulfate. The enzyme plays a crucial role in sulfate activation, the key step for sulfate utilization, and has been purified from crude extracts of Desulfovibrio desulfuricans ATCC 27774 and Desulfovibrio gigas. Both proteins are homotrimers [141 kDa (3 x 47) for D. desulfuricans and 147 kDa (3 x 49) for D. gigas] and have been identified, for the first time, as metalloproteins containing cobalt and zinc. EXAFS reveals that either cobalt or zinc binds endogenously at presumably equivalent metal binding sites and is tetrahedrally coordinated to one nitrogen and three sulfur atoms. Furthermore, the electronic absorption spectra display charge-transfer bands at 335 and 370 nm consistent with sulfur coordination to cobalt, and as expected for a distorted tetrahedral cobalt geometry, d-d bands are observed at 625, 666, and 715 nm. This geometry is supported by the observation of high-spin Co2+ EPR signals at g approximately 6.5.

Three-iron clusters in iron--sulfur proteins: An EPR study of the exchange interactions, Gayda, Jean-Pierre, Bertrand Patrick, Theodule Francois-Xavier, and Moura Jose J. G. , The Journal of Chemical Physics, Volume 77, Number 7, p.3387-3391, (1982) AbstractWebsite
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Observation of ligand-based redox chemistry at the active site of a molybdenum enzyme, George, G. N., Costa C., Moura J. J. G., and Moura I. , Journal of the American Chemical Society, Mar 24, Volume 121, Number 11, p.2625-2626, (1999) AbstractWebsite
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A novel protein-bound copper - Molybdenum cluster, George, G. N., Pickering I. J., Yu E. Y., Prince R. C., Bursakov S. A., Gavel O. Y., Moura I., and Moura J. J. G. , Journal of the American Chemical Society, Aug 30, Volume 122, Number 34, p.8321-8322, (2000) AbstractWebsite
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Activation of N2O reduction by the fully reduced micro4-sulfide bridged tetranuclear Cu Z cluster in nitrous oxide reductase, Ghosh, S., Gorelsky S. I., Chen P., Cabrito I., Moura J. J., Moura I., and Solomon E. I. , J Am Chem Soc, Dec 24, Volume 125, Number 51, p.15708-9, (2003) AbstractWebsite

The tetranuclear CuZ cluster catalyzes the two-electron reduction of N2O to N2 and H2O in the enzyme nitrous oxide reductase. This study shows that the fully reduced 4CuI form of the cluster correlates with the catalytic activity of the enzyme. This is the first demonstration that the S = 1/2 form of CuZ can be further reduced. Complementary DFT calculations support the experimental findings and demonstrate that N2O binding in a bent mu-1,3-bridging mode to the 4CuI form is most efficient due to strong back-bonding from two reduced copper atoms. This back-donation activates N2O for electrophilic attack by a proton.

Spectroscopic, computational, and kinetic studies of the mu4-sulfide-bridged tetranuclear CuZ cluster in N2O reductase: pH effect on the edge ligand and its contribution to reactivity, Ghosh, S., Gorelsky S. I., George S. D., Chan J. M., Cabrito I., Dooley D. M., Moura J. J., Moura I., and Solomon E. I. , J Am Chem Soc, Apr 4, Volume 129, Number 13, p.3955-65, (2007) AbstractWebsite

A combination of spectroscopy and density functional theory (DFT) calculations has been used to evaluate the pH effect at the CuZ site in Pseudomonas nautica (Pn) nitrous oxide reductase (N2OR) and Achromobacter cycloclastes (Ac) N2OR and its relevance to catalysis. Absorption, magnetic circular dichroism, and electron paramagnetic resonance with sulfur K-edge X-ray absorption spectra of the enzymes at high and low pH show minor changes. However, resonance Raman (rR) spectroscopy of PnN2OR at high pH shows that the 415 cm-1 Cu-S vibration (observed at low pH) shifts to higher frequency, loses intensity, and obtains a 9 cm-1 18O shift, implying significant Cu-O character, demonstrating the presence of a OH- ligand at the CuICuIV edge. From DFT calculations, protonation of either the OH- to H2O or the mu4-S2- to mu4-SH- would produce large spectral changes which are not observed. Alternatively, DFT calculations including a lysine residue at an H-bonding distance from the CuICuIV edge ligand show that the position of the OH- ligand depends on the protonation state of the lysine. This would change the coupling of the Cu-(OH) stretch with the Cu-S stretch, as observed in the rR spectrum. Thus, the observed pH effect (pKa approximately 9.2) likely reflects protonation equilibrium of the lysine residue, which would both raise E degrees and provide a proton for lowering the barrier for the N-O cleavage and for reduction of the [Cu4S(im)7OH]2+ to the fully reduced 4CuI active form for turnover.

The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase, Gilmour, R., Goodhew C. F., Pettigrew G. W., Prazeres S., Moura J. J., and Moura I. , Biochem J, Jun 15, Volume 300 ( Pt 3), p.907-14, (1994) AbstractWebsite

In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity ('turnover number') under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 min-1 and 13 microM. However, in this case, the kinetics deviate from first-order progress curves at all ferrocytochrome c concentrations. Consideration of the periplasmic environment in Paracoccus denitrificans leads us to propose that the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.

The affinity and specificity of Ca(2+)-binding sites of cytochrome-c peroxidase from Paracoccus denitrificans, Gilmour, R., Prazeres S., McGinnity D. F., Goodhew C. F., Moura J. J., Moura I., and Pettigrew G. W. , Eur J Biochem, Dec 15, Volume 234, Number 3, p.878-86, (1995) AbstractWebsite

The binding of Ca2+ to the dihaem cytochrome-c peroxidase from Paracoccus denitrificans was analysed by following perturbations in the visible and 1H-NMR spectra of both haem groups. The enzyme contains at least two types of Ca(2+)-binding site. Site I is occupied in the isolated enzyme, binds Ca2+ with a redox-state-independent Kd of 1.2 microM and accommodates neither Mg2+ nor Mn2+. Site II is unoccupied in dilute solutions of the isolated oxidised enzyme and binds Ca2+ cooperatively with a Kd of 0.52 mM. In the mixed valence form, the binding affinity increases to resemble that of site I. The cooperativity was shown by -Ca2+ binding to site II, the titration of haem methyl 1H-NMR resonances, and a half-of-sites effect observed for modification of an essential histidine with diethylpyrocarbonate. These are all consistent with site II being situated at the interface between two monomers of a dimeric enzyme. Thus the equilibrium of binding to site II is a reflection of the equilibrium for dimerisation and conditions which shift that equilibrium towards the dimer, such as increased ionic strength or high protein concentration, also increase Ca2+ affinity. Binding of Ca2+ to site II is required for formation of the active high spin state at the peroxidatic haem.

Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans, Gilmour, R., Goodhew C. F., Pettigrew G. W., Prazeres S., Moura I., and Moura J. J. , Biochem J, Sep 15, Volume 294 ( Pt 3), p.745-52, (1993) AbstractWebsite

The cytochrome c peroxidase of Paracoccus denitrificans is similar to the well-studied enzyme from Pseudomonas aeruginosa. Like the Pseudomonas enzyme, the Paracoccus peroxidase contains two haem c groups, one high potential and one low potential. The high-potential haem acts as a source of the second electron for H2O2 reduction, and the low-potential haem acts as a peroxidatic centre. Reduction with ascorbate of the high-potential haem of the Paracoccus enzyme results in a switch of the low-potential haem to a high-spin state, as shown by visible and n.m.r. spectroscopy. This high-spin haem of the mixed-valence enzyme is accessible to ligands and binds CN- with a KD of 5 microM. The Paracoccus enzyme is significantly different from that from Pseudomonas in the time course of high-spin formation after reduction of the high-potential haem, and in the requirement for bivalent cations. Reduction with 1 mM ascorbate at pH 6 is complete within 2 min, and this is followed by a slow appearance of the high-spin state with a half-time of 10 min. Thus the process of reduction and spin state change can be easily separated in time and the intermediate form obtained. This separation is also evident in e.p.r. spectra, although the slow change involves an alteration in the low-spin ligation at this temperature rather than a change in spin state. The separation is even more striking at pH 7.5, where no high-spin form is obtained until 1 mM Ca2+ is added to the mixed-valence enzyme. The spin-state switch of the low-potential haem shifts the midpoint redox potential of the high-potential haem by 50 mV, a further indication of haem-haem interaction.

Enzymatic spectrophotometric determination of nitrites in beer, Girotti, S., Ferri E. N., Fini F., Ruffini F., Budini R., Moura I., Almeida G., Costa C., Moura J. J. G., and Carrea G. , Analytical Letters, 1999, Volume 32, Number 11, p.2217-2227, (1999) AbstractWebsite

A colorimetric assay for nitrite determination in beer based on c-type multiheme enzyme Nitrite reductase (NiR) isolated from Desulfovibrio desulfuricans ATCC 27774, was developed. Using the enzyme in solution, nitrite assay was linear in the 10(-8) - 10(-2) M range with a detection limit of 10(-8) M. and a recovery ranging from 90 to 107%. The imprecision ranged from 4 to 10% on the entire calibration curve. With NIR immobilised onto a nylon coil, a flow reactor was developed which showed a narrower linear range (10(-5) - 10(-2) M) and a higher detection limit (10(-5) M) than with the enzyme in solution, but made it possible to reuse the enzyme up to 100 times (50% residual activity). Sample preparation was simple and fast: only degassing and beer dilution by buffer was needed. This enzymatic assay was in good agreement with the results obtained using commercial nitrite determination kits.

Protein effects on the electronic structure of the [Fe4S4]2+ cluster in ferredoxin and HiPIP, Glaser, T., Bertini I., Moura J. J., Hedman B., Hodgson K. O., and Solomon E. I. , J Am Chem Soc, May 23, Volume 123, Number 20, p.4859-60, (2001) AbstractWebsite
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Nitric oxide detection using electrochemical third-generation biosensors – based on heme proteins and porphyrins, Gomes, F., Maia L., Cordas C., Delerue-Matos C., Moura I., Moura J. J. G., and Morais S. , Electroanalysis, Volume 30, p.1-20, (2018) Website
Third-generation electrochemical biosensor based on nitric oxide reductase immobilized in a multiwalled carbon nanotubes/1-n-butyl-3-methylimidazolium tetrafluoroborate nanocomposite for nitric oxide detection, Gomes, FO, Maia L. B., Delerue-Matos C., Moura I., Moura J. J. G., and Morais S. , Sensors & Actuators: B. Chemical, Volume 285, p.445-452, (2019) Website
Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic bilayer nanocomposite, Gomes, FO, Maia L. B., Loureiro JA, Pereira MC, Delerue-Matos C., Moura I., Moura J. J. G., and Morais S. , Bioelectrochem, Volume 127, p.76-86, (2019)
Electroanalytical characterization of the direct Marinobacter hydrocarbonoclasticus nitric oxide reductase-catalysed nitric oxide and dioxygen reduction, Gomes, F., Maia L., Cordas C., Moura I., Delerue-Matos C., Moura J. J. G., and Morais S. , Bioelectrochem, Volume 125, p.8-14, (2019) Website