Cofactor dependence of reduction potentials for [4Fe-4S]2+/1+ in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine by a free radical mechanism. The 5'-deoxyadenosyl radical derived from the reductive cleavage of S-adenosyl-l-methionine (SAM) initiates substrate-radical formation. The [4Fe-4S](1+) cluster in LAM is the one-electron source in the reductive cleavage of SAM, which is directly ligated to the unique iron site in the cluster. We here report the midpoint reduction potentials of the [4Fe-4S](2+/1+) couple in the presence of SAM, S-adenosyl-l-homocysteine (SAH), or 5'-{N-[(3S)-3-aminocarboxypropyl]-N-methylamino}-5'-deoxyadenosine (azaSAM) as measured by spectroelectrochemistry. The reduction potentials are -430 +/- 2 mV in the presence of SAM, -460 +/- 3 mV in the presence of SAH, and -497 +/- 10 mV in the presence of azaSAM. In the absence of SAM or an analogue and the presence of dithiothreitol, dihydrolipoate, or cysteine as ligands to the unique iron, the midpoint potentials are -479 +/- 5, -516 +/- 5, and -484 +/- 3 mV, respectively. LAM is a member of the radical SAM superfamily of enzymes, in which the CxxxCxxC motif donates three thiolate ligands to iron in the [4Fe-4S] cluster and SAM donates the alpha-amino and alpha-carboxylate groups of the methionyl moiety as ligands to the fourth iron. The results show the reduction potentials in the midrange for ferredoxin-like [4Fe-4S] clusters. They show that SAM elevates the reduction potential by 86 mV relative to that of dihydrolipoate as the cluster ligand. This difference accounts for the SAM-dependent reduction of the [4Fe-4S](2+) cluster by dithionite reported earlier. Analogues of SAM have a weakened capacity to raise the potential. We conclude that the midpoint reduction potential of the cluster ligated to SAM is 1.2 V less negative than the half-wave potential for the one-electron reductive cleavage of simple alkylsulfonium ions in aqueous solution. The energetic barrier in the reductive cleavage of SAM may be overcome through the use of binding energy.     

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.     

Influence of the PsbA1/PsbA3, Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges on the redox potential of the primary quinone Q(A) in Photosystem II from Thermosynechococcus elongatus as revealed by spectroelectrochemistry

Ca(2+) and Cl(-) ions are essential elements for the oxygen evolution activity of photosystem II (PSII). It has been demonstrated that these ions can be exchanged with Sr(2+) and Br(-), respectively, and that these ion exchanges modify the kinetics of some electron transfer reactions at the Mn?Ca cluster level (Ishida et al., J. Biol. Chem. 283 (2008) 13330-13340). It has been proposed from thermoluminescence experiments that the kinetic effects arise, at least in part, from a decrease in the free energy level of the Mn(4)Ca cluster in the S? state though some changes on the acceptor side were also observed. Therefore, in the present work, by using thin-layer cell spectroelectrochemistry, the effects of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges on the redox potential of the primary quinone electron acceptor Q(A), E(m)(Q(A)/Q(A)(-)), were investigated. Since the previous studies on the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges were performed in PsbA3-containing PSII purified from the thermophilic cyanobacterium Thermosynechococcus elongatus, we first investigated the influences of the PsbA1/PsbA3 exchange on E(m)(Q(A)/Q(A)(-)). Here we show that i) the E(m)(Q(A)/Q(A)(-)) was up-shifted by ca. +38mV in PsbA3-PSII when compared to PsbA1-PSII and ii) the Ca(2+)/Sr(2+) exchange up-shifted the E(m)(Q(A)/Q(A)(-)) by ca. +27mV, whereas the Cl(-)/Br(-) exchange hardly influenced E(m)(Q(A)/Q(A)(-)). On the basis of the results of E(m)(Q(A)/Q(A)(-)) together with previous thermoluminescence measurements, the ion-exchange effects on the energetics in PSII are discussed.     

Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy

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.     

Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties

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.     

Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum

An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S.W., Lindahl, P.A., & Münck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (+/- 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (+/- 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy

Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD(+). The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 +/- 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) couples for the FMN (E(1) = -301 +/- 6 mV; E(2) = -252 +/- 8 mV) and FAD (E(1) = -312 +/- 6 mV; E(2) = -297 +/- 5 mV) were determined from analysis of spectral changes at 630 nm. Corresponding values for the midpoint reduction potentials for FMN (E(1) = -298 +/- 4 mV; E(2) = -259 +/- 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB. During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at approximately 630 nm, consistent with the measured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual signal is assigned to the formation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2S center. Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that are distinct from those produced by dithionite. From potentiometric studies, the reduction of the 2Fe-2S center and the reduction of the FMN occur concomitantly. The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulfur flavoprotein.     

Crystallographic analysis of the intact metal centres [3Fe-4S](1+/0) and [4Fe-4S](2+/1+) in a Zn(2+) -containing ferredoxin

Detailed structural models of di-cluster seven-iron ferredoxins constitute a valuable resource for folding and stability studies relating the metal cofactors' role in protein stability. The here reported, hemihedric twinned crystal structure at 2.0 A resolution from Acidianus ambivalens ferredoxin, shows an integral 103 residues, physiologically relevant native form composed by a N-terminal extension comprising a His/Asp Zn(2+) site and the ferredoxin (betaalphabeta)(2) core, which harbours intact clusters I and II, a [3Fe-4S](1+/0) and a [4Fe-4S](2+/1+) centres. This is in contrast with the previously available ferredoxin structure from Sulfolofus tokodai, which was obtained from an artificial oxidative conversion with two [3Fe-4S](1+/0) centres and poor definition around cluster II.     

Investigation of the environment surrounding iron-sulfur cluster 4 of Escherichia coli dimethylsulfoxide reductase

Iron-sulfur ([Fe-S]) clusters are common in electron transfer proteins, and their midpoint potentials (E(m) values) play a major role in defining the rate at which electrons are shuttled. The E(m) values of [Fe-S] clusters are largely dependent on the protein environment as well as solvent accessibility. The electron transfer subunit (DmsB) of Escherichia coli dimethylsulfoxide reductase contains four [4Fe-4S] clusters (FS1-FS4) with E(m) values between -50 and -330 mV. We have constructed an in silico model of DmsB and addressed the roles of a group of residues surrounding FS4 in electron transfer, menaquinol (MQH(2)) binding, and protein control of its E(m). Residues Pro80, Ser81, Cys102, and Tyr104 of DmsB are located at the DmsB-DmsC interface and are critical for the binding of the MQH(2) inhibitor analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) and the transfer of electrons from MQH(2) to FS4. Because the EPR spectrum of FS4 is complicated by spectral overlap and spin-spin interactions with the other [4Fe-4S] clusters of DmsB, we evaluated mutant effects on FS4 in double mutants (with a DmsB-C102S mutation) in which FS4 is assembled as a [3Fe-4S] cluster (FS4([3Fe)(-)(4S])). The DmsB-C102S/Y104D and DmsB-C102S/Y104E mutants dramatically lower the E(m) of FS4([3Fe)(-)(4S]) from 275 to 150 mV and from 275 to 145 mV, respectively. Mutations of positively charged residues around FS4([3Fe)(-)(4S]) lower its E(m), but mutations of negatively charged residues have negligible effects. The E(m) of FS4([3Fe)(-)(4S]) in the DmsB-C102S mutant is insensitive to HOQNO as well as to changes in pH from 5 to 7. The FS4([3Fe)(-)(4S]) E(m) of the DmsB-C102S/Y104D mutant increases in the presence of HOQNO and decreasing pH. Analyses of the mutants suggest that the maximum achievable E(m) for FS4([3Fe)(-)(4S]) of DmsB is approximately 275 mV.     

Biotin synthase contains two distinct iron-sulfur cluster binding sites: chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions.

Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Biotin synthase (BioB) is aerobically purified as a dimer that contains [2Fe-2S](2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing [4Fe-4S](2+) and/or [4Fe-4S](+) clusters. To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy. In contrast to previous reports, we find that aerobically purified BioB contains ca. 1.2-1.5 [2Fe-2S](2+) clusters per monomer with epsilon(452) = 8400 M(-)(1) cm(-)(1) per monomer. Upon reduction, the [2Fe-2S](2+) clusters are converted to [4Fe-4S] clusters with two widely separate reduction potentials of -140 and -430 mV. BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two [4Fe-4S](2+) clusters per monomer with epsilon(400) = 30 000 M(-)(1) cm(-)(1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively. Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two [2Fe-2S](2+) and two [4Fe-4S](2+) clusters.     

The effect of substrate, dihydrobiopterin, and dopamine on the EPR spectroscopic properties and the midpoint potential of the catalytic iron in recombinant human phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH(4)) and non-heme iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic ferric iron at the active site of recombinant human PAH (hPAH) and its midpoint potential at pH 7.25 (E(m)(Fe(III)/Fe(II))) were studied by EPR spectroscopy. Similar EPR spectra were obtained for the tetrameric wild-type (wt-hPAH) and the dimeric truncated hPAH(Gly(103)-Gln(428)) corresponding to the "catalytic domain." A rhombic high spin Fe(III) signal with a g value of 4.3 dominates the EPR spectra at 3.6 K of both enzyme forms. An E(m) = +207 +/- 10 mV was measured for the iron in wt-hPAH, which seems to be adequate for a thermodynamically feasible electron transfer from BH(4) (E(m) (quinonoid-BH(2)/BH(4)) = +174 mV). The broad EPR features from g = 9.7-4.3 in the spectra of the ligand-free enzyme decreased in intensity upon the addition of L-Phe, whereas more axial type signals were observed upon binding of 7,8-dihydrobiopterin (BH(2)), the stable oxidized form of BH(4), and of dopamine. All three ligands induced a decrease in the E(m) value of the iron to +123 +/- 4 mV (L-Phe), +110 +/- 20 mV (BH(2)), and -8 +/- 9 mV (dopamine). On the basis of these data we have calculated that the binding affinities of L-Phe, BH(2), and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme, with respect to the ferric form. Interestingly, an E(m) value comparable with that of the ligand-free, resting form of wt-hPAH, i.e. +191 +/- 11 mV, was measured upon the simultaneous binding of both L-Phe and BH(2), representing an inactive model for the iron environment under turnover conditions. Our findings provide new information on the redox properties of the active site iron relevant for the understanding of the reductive activation of the enzyme and the catalytic mechanism.     

Sulfur dehydrogenase of Paracoccus pantotrophus: the heme-2 domain of the molybdoprotein cytochrome c complex is dispensable for catalytic activity

Sulfur dehydrogenase, Sox(CD)(2), is an essential part of the sulfur-oxidizing enzyme system of the chemotrophic bacterium Paracoccus pantotrophus. Sox(CD)(2) is a alpha(2)beta(2) complex composed of the molybdoprotein SoxC (43 442 Da) and the hybrid diheme c-type cytochrome SoxD (37 637 Da). Sox(CD)(2) catalyzes the oxidation of protein-bound sulfur to sulfate with a unique six-electron transfer. Amino acid sequence analysis identified the heme-1 domain of SoxD proteins to be specific for sulfur dehydrogenases and to contain a novel ProCysMetXaaAspCys motif, while the heme-2 domain is related to various cytochromes c(2). Purification of sulfur dehydrogenase without protease inhibitor yielded a dimeric SoxCD(1) complex consisting of SoxC and SoxD(1) of 30 kDa, which contained only the heme-1 domain. The heme-2 domain was isolated as a new cytochrome SoxD(2) of about 13 kDa. Both hemes of SoxD in Sox(CD)(2) are redox-active with midpoint potentials at E(m)1 = 218 +/- 10 mV and E(m)2 = 268 +/- 10 mV, while SoxCD(1) and SoxD(2) both exhibit a midpoint potential of E(m) = 278 +/- 10 mV. Electrochemically induced FTIR difference spectra of Sox(CD)(2), SoxCD(1), and SoxD(2) were distinct. A carboxy group is protonated upon reduction of the SoxD(1) heme but not for SoxD(2). The specific activity of SoxCD(1) and Sox(CD)(2) was identical as was the yield of electrons with thiosulfate in the reconstituted Sox enzyme system. To examine the physiological significance of the heme-2 domain, a mutant was constructed that was deleted for the heme-2 domain, which produced SoxCD(1) and transferred electrons from thiosulfate to oxygen. These data demonstrated the crucial role of the heme-1 domain of SoxD for catalytic activity, electron yield, and transfer of the electrons to the cytoplasmic membrane, while the heme-2 domain mediated the alpha(2)beta(2) tetrameric structure of sulfur dehydrogenase.     

Effect of serinate ligation at each of the iron sites of the [Fe4S4] cluster of Pyrococcus furiosus ferredoxin on the redox, spectroscopic, and biological properties.

Pyrococcus furiosus ferredoxin (Fd) contains a single [Fe(4)S(4)] cluster coordinated by three cysteine (at positions 11, 17, and 56) and one aspartate ligand (at position 14). In this study, the spectroscopic, redox, and functional consequences of D14C, D14C/C11S, D14S, D14C/C17S, and D14C/C56S mutations have been investigated. The four serine variants each contain a potential cluster coordination sphere of one serine and three cysteine residues, with serine ligation at each of the four Fe sites of the [Fe(4)S(4)] cluster. All five variants were expressed in Escherichia coli, and each contained a [Fe(4)S(4)](2+,+) cluster as shown by UV-visible absorption and resonance Raman studies of the oxidized protein and EPR and variable-temperature magnetic circular dichroism (VTMCD) studies of the as-prepared, dithionite-reduced protein. Changes in both the absorption and resonance Raman spectra are consistent with changing from complete cysteinyl cluster ligation in the D14C variant to three cysteines and one oxygenic ligand in each of the four serine variants. EPR and VTMCD studies show distinctive ground and excited state properties for the paramagnetic [Fe(4)S(4)](+) centers in each of these variant proteins, with the D14C and D14C/C11S variants having homogeneous S = (1)/(2) ground states and the D14S, D14C/C17S, and D14C/C56S variants having mixed-spin, S = (1)/(2) and (3)/(2) ground states. The midpoint potentials (pH 7.0, 23 degrees C) of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (E(m) = -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were more negative by 49 and 78 mV, respectively. Since the VTMCD spectra indicate the presence of a valence-delocalized Fe(2. 5+)Fe(2.5+) pair in all five variants, the midpoint potentials are interpreted in terms of Cys11 and Cys17 ligating the nonreducible valence-delocalized pair in D14C. Only the D14S variant exhibited a pH-dependent redox potential over the range of 3.5-10, and this is attributed to protonation of the serinate ligand to the reduced cluster (pK(a) = 4.75). All five variants had similar K(m) and V(m) values in a coupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and oxidized by ferredoxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, the mode of ligation at each Fe atom in the [Fe(4)S(4)] cluster appears to have little effect on the interaction and the electron transfer between Fd and FNOR.     

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: purification and characterization

Dicamba O-demethylase is a multicomponent enzyme that catalyzes the conversion of the herbicide 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to 3,6-dichlorosalicylic acid (DCSA). The three components of the enzyme were purified and characterized. Oxygenase(DIC) is a homotrimer (alpha)3 with a subunit molecular mass of approximately 40 kDa. FerredoxinDIC and reductaseDIC are monomers with molecular weights of approximately 14 and 45 kDa, respectively. EPR spectroscopic analysis suggested the presence of a single [2Fe-2S](2+/1+) cluster in ferredoxinDIC and a single Rieske [2Fe-2S](2+; 1+) cluster within oxygenaseDIC. Consistent with the presence of a Rieske iron-sulfur cluster, oxygenaseDIC displayed a high reduction potential of E(m,7.0) = -21 mV whereas ferredoxinDIC exhibited a reduction potential of approximately E(m,7.0) = -171 mV. Optimal oxygenaseDIC activity in vitro depended on the addition of Fe2+. The identification of formaldehyde and DCSA as reaction products demonstrated that dicamba O-demethylase acts as a monooxygenase. Taken together, these data suggest that oxygenaseDIC is an important new member of the Rieske non-heme iron family of oxygenases.     

Oxidation of the non-heme iron complex in photosystem II

In photosystem II (PSII), the redox properties of the non-heme iron complex (Fe complex) are sensitive to the redox state of quinones (Q(A/)(B)), which may relate to the electron/proton transfer. We calculated the redox potentials for one-electron oxidation of the Fe complex in PSII [E(m)(Fe)] based on the reference value E(m)(Fe) = +400 mV at pH 7 in the Q(A)(0)Q(B)(0) state, considering the protein environment in atomic detail and the associated changes in protonation pattern. Our model yields the pH dependence of E(m)(Fe) with -60 mV/pH as observed in experimental redox titration. We observed significant deprotonation at D1-Glu244 in the hydrophilic loop region upon Fe complex oxidation. The calculated pK(a) value for D1-Glu244 depends on the Fe complex redox state, yielding a pK(a) of 7.5 and 5.5 for Fe(2+) and Fe(3+), respectively. To account for the pH dependence of E(m)(Fe), a model involving not only D1-Glu244 but also the other titratable residues (five Glu in the D-de loops and six basic residues near the Fe complex) seems to be needed, implying the existence of a network of residues serving as an internal proton reservoir. Reduction of Q(A/B) yields +302 mV and +268 mV for E(m)(Fe) in the Q(A)(-)Q(B)(0) and Q(A)(0)Q(B)(-) states, respectively. Upon formation of the Q(A)(0)Q(B)(-) state, D1-His252 becomes protonated. Forming Fe(3+)Q(B)H(2) by a proton-coupled electron transfer process from the initial state Fe(2+)Q(B)(-) results in deprotonation of D1-His252. The two EPR signals observed at g = 1.82 and g = 1.9 in the Fe(2+)Q(A)(-) state of PSII may be attributed to D1-His252 with variable and fixed protonation, respectively.     

DNA-bound redox activity of DNA repair glycosylases containing [4Fe-4S] clusters

MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.     

Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. M?ssbauer and EPR characterization of the metal centers

The hydrogenase (EC 1.2.2.1) 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 M?ssbauer 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.     

Patch-clamp recording of charge movement, Ca(2+) current, and Ca(2+) transients in adult skeletal muscle fibers

Intramembrane charge movement (Q), Ca(2+) conductance (G(m)) through the dihydropyridine-sensitive L-type Ca(2+) channel (DHPR) and intracellular Ca(2+) fluorescence (F) have been recorded simultaneously in flexor digitorum brevis muscle fibers of adult mice, using the whole-cell configuration of the patch-clamp technique. The voltage distribution of Q was fitted to a Boltzmann equation; the Q(max), V(1/2Q), and effective valence (z(Q)) values were 41 +/- 3.1 nC/&mgr;F, -17.6 +/- 0.7 mV, and 2.0 +/- 0.12, respectively. V(1/2G) and z(G) values were -0.3 +/- 0.06 mV and 5.6 +/- 0.34, respectively. Peak Ca(2+) transients did not change significantly after 30 min of recording. F was fit to a Boltzmann equation, and the values for V(F1/2) and z(F) were 6.2 +/- 0.04 mV and 2.4, respectively. F was adequately fit to the fourth power of Q. These results demonstrate that the patch-clamp technique is appropriate for recording Q, G(m), and intracellular [Ca(2+)] simultaneously in mature skeletal muscle fibers and that the voltage distribution of the changes in intracellular Ca(2+) can be predicted by a Hodgkin-Huxley model.