Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched 61Ni and 57Fe media has been purified to apparent homogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. they were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase II), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogenase II was selected for EPR and Mossbauer studies. As isolated, hydrogenase II exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of 61Ni proves that the rhombic signal is due to Ni. Combining the Mossbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the Mossbauer data also revealed two [4Fe-4S]2+ clusters iun each molecule of hydrogenase II. The EPR and Mossbauer data of hydrogenase I were found to be identical to those of hydrogenase II, indicating that both enzymes have common metallic centers.

The di-haem cytochrome c peroxidase of Paracoccus denitrificans is a calcium binding dimer of 37.5 kDa subunits. It is responsible for reduction of H(2)O(2) to H(2)O with oxidation of cytochrome c(550) and is isolated in a fully oxidised state (inactive) in which one haem (centre I) is in a high-spin/low-spin equilibrium and high potential and the other (centre II) is low-spin and low potential. The enzyme undergoes direct electron transfer (without the need for mediators) with a 4,4'-dithiodipyridine-modified gold electrode and the response of both haem groups can be observed. By combination of the cyclic and pulse voltammetric data with the established spectroscopic information, it was demonstrated that entry of one electron to the high potential haem leads (in a mechanism involving strong haem-haem interactions) to a complex change of spin states and redox potentials of both haems in order to attain a "ready state" for binding, reduction and cleavage of the hydrogen peroxide. In the absence of endogenous calcium, haem communication can be completely disconnected and is recovered only when Ca(2+) is added, an essential step for the formation of the peroxidatic site. The intricate electrochemical behaviour of this enzyme was interpreted as a mechanism involving, both reduction and oxidation of the high potential haem, an interfacial electron transfer coupled to a homogenous chemical reaction (EC mechanism). We discuss two different models for the sequence of events leading to the appearance of the active pentacoordinated peroxidatic haem.

The biological relevance of molybdenum was demonstrated in the early 1950s-1960s, by Bray, Beinert, Lowe, Massey, Palmer, Ehrenberg, Pettersson, Vänngård, Hanson and others, with ground-breaking studies performed, precisely, by electron paramagnetic resonance (EPR) spectroscopy. Those earlier studies, aimed to investigate the mammalian xanthine oxidase and avian sulfite oxidase enzymes, demonstrated the surprising biological reduction of molybdenum to the paramagnetic Mo⁵⁺. Since then, EPR spectroscopy, alongside with other spectroscopic methods and X-ray crystallography, has contributed to our present detailed knowledge about the active site structures, catalytic mechanisms and structure/activity relationships of the molybdenum-containing enzymes.
This Chapter will provide a perspective on the contribution that EPR spectroscopy has made to some selected systems. After a brief overview on molybdoenzymes, the Chapter will be focused on the EPR studies of mammalian xanthine oxidase, with a brief account on the prokaryotic aldehyde oxidoreductase, nicotinate dehydrogenase and carbon monoxide dehydrogenase, vertebrate sulfite oxidase, and prokaryotic formate dehydrogenases and nitrate reductases.

The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 10(6)-10(8) M-1 s-1 and increase in the direction D. desulfuricans cytochrome c3-->D. vulgaris cytochrome c3-->methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved. Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility). Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported.

Formate dehydrogenases (FDHs) are enzymes that catalyze the formate oxidation to carbon dioxide and that contain either Mo or W in a mononuclear form in the active site. In the present work, the influence of Mo and W salts on the production of FDH by Desulfovibrio alaskensis NCIMB 13491 was studied. Two different FDHs, one containing W (W-FDH) and a second incorporating either Mo or W (Mo/W-FDH), were purified. Both enzymes were isolated from cells grown in a medium supplemented with 1 muM molybdate, whereas only the W-FDH was purified from cells cultured in medium supplemented with 10 muM tungstate. We demonstrated that the genes encoding the Mo/W-FDH are strongly downregulated by W and slightly upregulated by Mo. Metal effects on the expression level of the genes encoding the W-FDH were less significant. Furthermore, the expression levels of the genes encoding proteins involved in molybdate and tungstate transport are downregulated under the experimental conditions evaluated in this work. The molecular and biochemical properties of these enzymes and the selective incorporation of either Mo or W are discussed.

Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite reductase, cytochrome c peroxidase and cytochrome cd1 nitrite reductase), non-heme proteins (superoxide reductase and [NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion.

Small electron-transfer proteins such as flavodoxin (16 kDa) and the tetraheme cytochrome c3 (13 kDa) have been used to mimic, in vitro, part of the complex electron-transfer chain operating between substrate electron donors and respiratory electron acceptors, in sulfate-reducing bacteria (Desulfovibrio species). The nature and properties of the complex formed between these proteins are revealed by 1H-NMR and molecular modeling approaches. Our previous study with the Desulfovibrio vulgaris proteins [Moura, I., Moura, J.J. G., Santos, M.H., & Xavier, A. V. (1980) Cienc. Biol. (Portugal) 5, 195-197; Stewart, D.E. LeGall, J., Moura, I., Moura, J. J. G., Peck, H.D. Jr., Xavier, A. V., Weiner, P. K., & Wampler, J.E. (1988) Biochemistry 27, 2444-2450] indicated that the complex between cytochrome c3 and flavodoxin could be monitored by changes in the NMR signals of the heme methyl groups of the cytochrome and that the electrostatic surface charge (Coulomb's law) on the two proteins favored interaction between one unique heme of the cytochrome with flavodoxin. If the interaction is indeed driven by the electrostatic complementarity between the acidic flavodoxin and a unique positive region of the cytochrome c3, other homologous proteins from these two families of proteins might be expected to interact similarly. In this study, three homologous Desulfovibrio cytochromes c3 were used, which show a remarkable variation in their individual isoelectric points (ranging from 5.5 to 9.5). On the basis of data obtained from protein-protein titrations followed at specific proton NMR signals (i.e., heme methyl resonances), a binding model for this complex has been developed with evaluation of stoichiometry and binding constants. This binding model involves one site on the cytochromes c3 and two sites on the flavodoxin, with formation of a ternary complex at saturation. In order to understand the potential chemical form of the binding model, a structural model for the hypothetical ternary complex, formed between one molecule of Desulfovibrio salexigens flavodoxin and two molecules of cytochrome c3, is proposed. These molecular models of the complexes were constructed on the basis of complementarity of Coulombic electrostatic surface potentials, using the available X-ray structures of the isolated proteins and, when required, model structures (D. salexigens flavodoxin and Desulfovibrio desulfuricans ATCC 27774 cytochrome c3) predicted by homology modeling.

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) belongs to the category of [Fe] hydrogenase which contains only iron-sulfur clusters as its prosthetic groups. Amino acid analyses were performed on the purified D. vulgaris hydrogenase. The amino acid composition obtained compared very well with the result derived from the nucleotide sequence of the structural gene (Voordouw, G., Brenner, S. (1985) Eur. J. Biochem. 148, 515-520). Detailed EPR reductive titration studies on the D. vulgaris hydrogenase were performed to characterize the metal centers in this hydrogenase. In addition to the three previously observed EPR signals (namely, the "isotropic" 2.02 signal, the rhombic 2.10 signal, and the complex signal of the reduced enzyme), a rhombic signal with resonances at the g-values of 2.06, 1.96, and 1.89 (the rhombic 2.06 signal) was detected when the samples were poised at potentials between 0 and -250 mV (with respect to normal hydrogen electrode). The midpoint redox potentials for each of the four EPR-active species were determined, and the characteristics of each EPR signal are described. Both the rhombic 2.10 and 2.06 signals exhibit spectral properties that are distinct from a ferredoxin-type [4Fe-4S] cluster and are proposed to originate from the same H2-binding center but in two different conformations. The complex signal of the reduced hydrogenase has been shown to represent two spin-spin interacting ferredoxin-type [4Fe-4S]1+ clusters (Grande, H. J., Dunham, W. R., Averill, B., Van Dijk, C., and Sands, R. H. (1983) Eur. J. Biochem. 136, 201-207). The titration data indicated a strong cooperative effect between these two clusters during their reduction. In an effort to accurately estimate the number of iron atoms/molecule of hydrogenase, plasma emission and chemical methods were used to determine the iron contents in the samples; and four different methods, including amino acid analysis, were used for protein determination. The resulting iron stoichiometries were found to be method-dependent and vary over a wide range (+/- 20%). The uncertainties involved in the determination of iron stoichiometry are discussed.

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.

The EPR characterization of the molybdenum(V) forms obtained on formate reduction of both as-prepared and inhibited formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774, an enzyme that catalyzes the oxidation of formate to CO(2), is reported. The Mo(V) EPR signal of the as-prepared formate-reduced enzyme is rhombic (g(max)=2.012, g(mid)=1.996, g(min)=1.985) and shows hyperfine coupling with two nuclear species with I=1/2. One of them gives an anisotropic splitting and is not solvent exchangeable (A(max)=11.7, A(mid)=A(min)=non-detectable, A-values in cm(-1)x10(-4)). The second species is exchangeable with solvent and produces a splitting at the three principal g-values (A(max)=7.7, A(mid)=10.0, A(min)=9.3). The hyperfine couplings of the non-solvent and solvent exchangeable nuclei are assigned to the hydrogen atoms of the beta-methylene carbon of a selenocysteine and to a Mo ligand whose nature, sulfydryl or hydroxyl, is still in debate. The Mo(V) species obtained in the presence of inhibitors (azide or cyanide) yields a nearly axial EPR signal showing only one detectable splitting given by nuclear species with I=1/2 (g(max)=2.092, g(mid)=2.000, g(min)=1.989, A(max)=non-detectable, A(mid)=A(min)=7.0), which is originated from the alpha-proton donated by the formate to a proximal ligand of the molybdenum. The possible structures of both paramagnetic molybdenum species (observed upon formate reduction in presence and absence of inhibitors) are discussed in comparison with the available structural information of this enzyme and the structural and EPR properties of the closely related formate dehydrogenase-H from Escherichia coli.

Iron-sulfur clusters with [3Fe-4S] cores are widely distributed in biological systems. In the oxidized state, designated [3Fe-4S](+), these electron-transfer agents have an electronic ground state with S = 1/2, and; they exhibit EPR signals centered at g = 2.01. It has been established by Mossbauer spectroscopy that the three iron sites of the cluster are high-spin Fe3+; and the general properties of the S = 1/2 ground state have been described with the exchange Hamiltonian H-exch = J(12)S(1).S-2 + J(23)S(2).S-3 + J(13)S(1).S-3 Some [3Fe-4S](+) clusters (type 1) have their g-values confined to the range between g = 2.03 and 2.00 while others (type 2) exhibit a continuous distribution of g-values down to g approximate to 1.85. Despite considerable efforts in various laboratories no model has emerged that explains the g-values of type 2 clusters. The 4.2 K spectra of all [3Fe-4S](+) clusters have broad features,which have been simulated in the past by using Fe-57 magnetic hyperfine tensors with anisotropies that are unusually large for high-spin feme sites. It is proposed here that antisymmetric exchange, H-AS = d.(S-1 x S-2 + S-2 x S-3 + S-3 x S-1), is the cause of the g-value shifts in type 2 clusters. We have been able to fit the EPR and Mossbauer spectra of the 3Fe clusters of beef heart aconitase and Desulfovibrio gigas ferredoxin II by using antisymmetric exchange in combination with distributed exchange coupling constants J(12), J(13), and J(23) (J-strain). While antisymmetric exchange is negligible for aconitase (which has a type 1 cluster), fits of the ferredoxin II spectra require \d\ approximate to 0.4 cm(-1). Our studies show that the data of both proteins can lie fit using the same isotropic Fe-57 magnetic hyperfine coupling constant for th three cluster sites, namely a -18.0 MHz for aconitase and a = -18.5 MHz for the D. gigas ferredoxin. The effects of antisymmetric exchange and J-strain on the Mossbauer and EPR spectra are discussed.

Membrane electrodes (ME) were constructed using gold, glassy carbon and pyrolytic graphite supports and a dialysis membrane, and used to study the electrochemical behavior of small size electron transfer proteins: monohemic cytochrome c(522) from Pseudomonas nautica and cytochrome c(533) as well as rubredoxin from Desulfovibrio vulgaris. Different electrochemical techniques were used including cyclic voltammetry (CV), square wave voltammetry (SW) and differential pulse voltammetry (DP). A direct electrochemical response was obtained in all cases except with rubredoxin where a facilitator was added to the protein solution entrapped between the membrane and the electrode surface. Formal potentials and heterogeneous charge transfer rate constants were determined from the voltammetric data. The influence of the ionic strength and the pH of the medium on the electrochemical response at the ME were analyzed. The benefits from the use of the ME in protein electrochemistry and its role in modulating the redox behavior are analyzed. A critical comparison is presented with data obtained at non-MEs. Finally, the interactions that must be established between the proteins and the electrode surfaces are discussed, thereby modeling molecular interactions that occur in biological systems. (C) 2002 Elsevier Science B.V. All rights reserved.

A c-type hexaheme nitrite reductase (NiR) isolated from nitrate-grown cells of Desulfovibrio desulfuricans (Dd) ATCC 27774 catalyses the six-electron reduction of nitrite to ammonia. Previous electrochemical studies demonstrated that a simple electrocatalytic mechanism can be applied to this system (Moreno, C., Costa, C., Moura, I., LeGall, J., Liu, M. Y., Payne, W. J., Van Dijk, C. and Moura, J. J. G. (1992) Eur.J.Biochem. 212, 79-86). Its substrate specificity, availability and stability under ambient conditions makes this enzymatic system a promising candidate for use in a biosensor device. An electrochemical study of gel-immobilized Dd NiR on a glassy carbon electrode revealed both enzymatic activity and amperometric response to nitrite. In this study it was observed that the catalytic current density is a function of the nitrite concentration in solution and follows a characteristic Michaelis-Menten-type substrate dependence. Such a biosensor device (NiR-electrode) bears the option to be used for analytical determination of nitrite in complex media.

In this paper we propose the construction of a new non-mediated electrochemical biosensor for nitrite determination in complex samples. The device is based on the stable and selective cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans, which has both high turnover and heterogeneous electron transfer rates. In opposition to previous efforts making use of several redox mediators, in this work we exploited the capacity of ccNiR to display a direct electrochemical response when interacting with pyrolytic graphite (PG) surfaces. To enable the analytical application of such bioelectrode the protein was successfully incorporated within a porous silica glass made by the sol-gel process. In the presence of nitrite, the ccNiR/sol-gel/PG electrode promptly displays catalytic currents indicating that the entrapped ccNiR molecules are reduced via direct electron transfer. This result is noteworthy since the protein molecules are caged inside a non-conductive silica network, in the absence of any mediator species or electron relay. At optimal conditions, the minimum detectable concentration is 120 nM. The biosensor sensitivity is 430 mA M(-1) cm(-2) within a linear range of 0.25-50 microM, keeping a stable response up to two weeks. The analysis of nitrites in freshwaters using the method of standard addition was highly accurated.

Single-walled carbon nanotubes (SWCNTs) deposits on glassy carbon and pyrolytic graphite electrodes have dramatically enhanced the direct electron transfer of the multihemic nitrite reductase from Desulfovibrio desulfuricans ATCC 27774, enabling a 10-fold increase in catalytic currents. At optimal conditions, the sensitivity to nitrite and the maximum current density were 2.4 +/- 0.1 A L mol(-1) cm(-2) and 1500 mu A cm(-2), respectively. Since the biosensor performance decreased over time, laponite clay and electropolymerized amphiphilic pyrrole were tested as protecting layers. Both coating materials increased substantially the bioelectrode stability, which kept about 90% and 60% of its initial sensitivity to nitrite after 20 and 248 days, respectively.

A hypothetical model of the complex formed between the iron-sulfur protein rubredoxin and the tetraheme cytochrome c3 from the sulfate-reducing bacteria Desulfovibrio vulgaris (Hildenborough) has been proposed utilizing computer graphic modeling, computational methods and NMR spectroscopy. The proposed complex appears feasible on the basis of complementary electrostatic interaction and steric factors and is consistent with the data from NMR experiments. In this model, the non-heme iron atom of rubredoxin is in close proximity to heme 1 of cytochrome c3. The complex is stabilized by charge-pair interactions and hydrogen bonds. This complex is compared to the flavodoxin-cytochrome c3 complex previously proposed [Stewart, D. E., LeGall, J., Moura, I., Moura, J. J. G., Peck, H. D. Jr, Xavier, A. V., Weiner, P. K. & Wampler, J. E. (1988) Biochemistry 27, 2444-2450] and new NMR data shows that both proteins interact with the same heme group of the cytochrome as postulated.

Desulfovibrio gigas hydrogenase (EC 1.12.2.1) is a complex enzyme containing one nickel, one 3Fe, and two [Fe4S4] clusters (Teixeira, M., Moura, I., Xavier, A. V., Der Vartanian, D. V., LeGall, J., Peck, H. D., Jr., Huynh, B. H., and Moura, J. J. G. (1983) Eur. J. Biochem. 130, 481-484). This hydrogenase belongs to a class of enzymes that are inactive "as isolated" (the so-called "oxygen-stable hydrogenases") and must go through an activation process in order to express full activity. The state of characterization of the active centers of the enzyme as isolated prompted us to do a detailed analysis of the redox patterns, activation profile, and catalytic redox cycle of the enzyme in the presence of either the natural substrate (H2) or chemical reductants. The effect of natural cofactors, as cytochrome C3, was also studied. Special focus was given to the intermediate redox species generated during the catalytic cycle of the enzyme and to the midpoint redox potentials associated. The available information is discussed in terms of a "working hypothesis" for the mechanism of the [NiFe] hydrogenases from sulfate reducing organisms in the context of activation process and catalytic cycle.