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ABSTRACT: Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe-4S]1+, [4Fe-4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA-SerB-SerC, a hetero-trimetric complex of alphabetagamma subunits) revealed that the [3Fe-4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (E(m)) of +118+/-10 mV for the [3Fe-4S]1+/0 couple. A [4Fe-4S]1+ cluster EPR signal developed over a range of potentials between 300 and -200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183+/-10 mV (FS1) and -51+/-10 mV (FS3) for the two [4Fe-4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe-4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (E(m)) of about -356 mV (FS2). It is considered likely that, at low redox potential (E(m) less than -300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe-4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe-4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3 approximately 1.999, g2 approximately 1.996 and g1 approximately 1.965 (g(av) 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g approximately 3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.
Biochemical Journal 12/2007; 408(1):19-28. · 4.90 Impact Factor
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ABSTRACT: The Escherichia coli NapA (periplasmic nitrate reductase) contains a [4Fe-4S] cluster and a Mo-bis-molybdopterin guanine dinucleotide cofactor. The NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). These proteins have been purified and studied by spectropotentiometry, and the structure of NapA has been determined. In contrast to the well characterized heterodimeric NapAB systems ofalpha-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the gamma-proteobacterial E. coli NapA and NapB proteins purify independently and not as a tight heterodimeric complex. This relatively weak interaction is reflected in dissociation constants of 15 and 32 mum determined for oxidized and reduced NapAB complexes, respectively. The surface electrostatic potential of E. coli NapA in the apparent NapB binding region is markedly less polar and anionic than that of the alpha-proteobacterial NapA, which may underlie the weaker binding of NapB. The molybdenum ion coordination sphere of E. coli NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 A, which is indicative of a water ligand. The potential range over which the Mo(6+) state is reduced to the Mo(5+) state in either NapA (between +100 and -100 mV) or the NapAB complex (-150 to -350 mV) is much lower than that reported for R. sphaeroides NapA (midpoint potential Mo(6+/5+) > +350 mV), and the form of the Mo(5+) EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo(5+) state could not be further reduced to Mo(4+). We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo(5+) ion and where a stable des-oxo Mo(6+) species may participate.
Journal of Biological Chemistry 04/2007; 282(9):6425-37. · 4.77 Impact Factor
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ABSTRACT: Protein film voltammetry of Paracoccus pantotrophus respiratory nitrate reductase (NarGH) and Synechococcus elongatus assimilatory nitrate reductase (NarB) shows that reductive activation of these enzymes may be required before steady state catalysis is observed. For NarGH complementary spectroscopic studies suggest a structural context for the activation. Catalytic protein film voltammetry at a range of temperatures has allowed quantitation of the activation energies for nitrate reduction. For NarGH with an operating potential of ca. 0.05 V the activation energy of ca. 35 kJ mol-1 is over twice that measured for NarB whose operating potential is ca. -0.35 V.
Dalton Transactions 12/2005; · 3.84 Impact Factor
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ABSTRACT: Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S](1+) cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly [4Fe-4S](1+) and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.
Journal of Biological Chemistry 08/2004; 279(31):32212-8. · 4.77 Impact Factor
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ABSTRACT: Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate
reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed
nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature
EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S]1+ cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (Em) of –190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed “very high g” with gav = 2.0047 in air-oxidized enzyme that accounted for only 10–20% of the total molybdenum. This signal disappeared upon reduction
with dithionite, and a new “high g” species (gav = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of –100 to
–350 mV (Em Mo6+/5+ = –150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed
that activity is turned on at potentials below –200 mV, where the cofactors are predominantly [4Fe-4S]1+ and Mo5+. The data suggests that during the catalytic cycle nitrate will bind to the Mo5+ state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those
of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic
enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling
of NarB to low potential ferredoxins in the cell cytoplasm.
Journal of Biological Chemistry 07/2004; 279(31):32212-32218. · 4.77 Impact Factor
