Iron-sulfur cluster biosynthesis. Kinetic analysis of [2Fe-2S] cluster transfer from holo ISU to apo Fd: role of redox chemistry and a conserved aspartate

ISU-type proteins mediate cluster transfer to apo protein targets. Rate constants have been determined for cluster transfer from ISU to apo Fd for both Homo sapiens and Schizosaccharomyces pombe proteins, and cross reactions have also been examined. Substitution of a key aspartate residue of ISU is found to decrease the rate of cluster transfer by at least an order of magnitude (for wild-type Hs ISU cluster transfer to Hs apo Fd, k(2) approximately 540 M(-1) min(-1), relative 56 M(-1) min(-1) for D37A ISU). This change in rate constant does not reflect any change in binding affinity of the ISU and Fd proteins. The pH dependencies of cluster transfer rates are similar for WT and D37A ISU, arguing against a role for Asp37 as a catalytic base, although evidence for general base catalysis mediating deprotonation of Cys from the apo target is supported by an observed pK(a) of 6.9 determined from the pH profiles for both WT and D37A ISU. Such a pK(a) value is at the lower limit for Cys and is common for solvent-accessible Cys thiols. The temperature dependence of the rate constant defining the cluster transfer reaction for wild type versus the aspartate derivative is distinct. Thermal activation parameters (DeltaH and DeltaS) are consistent with a solvent-accessible ISU-bound cluster, with desolvation as a principle barrier to cluster transfer. Experiments to determine the dependence of reaction rate constants on viscosity indicate cluster transfer to be rate-limiting. Fully oxidized cluster appears to be the natural state for transfer to target proteins. Reduced Fd does not readily reduce ISU-bound [2Fe-2S](2+) and does not promote cluster transfer to an apo Fd target.     

Iron-sulfur cluster biosynthesis. A comparative kinetic analysis of native and Cys-substituted ISA-mediated [2Fe-2S]2+ cluster transfer to an apoferredoxin target

ISA type proteins mediate cluster transfer to apoprotein targets. Rate constants have been determined for cluster transfer from Schizosaccharomyces pombe ISA to apo Fd. Substitution of the cysteine residues of ISA produced derivative proteins (C72A, C136A, and C138A) that were found to be at least as active in cluster transfer reactions as the native form at 25 degrees C (k(2) approximately 170 M(-1) min(-1) for native, k(2) approximately 169 M(-1) min(-1) for C72A, k(2) approximately 206 M(-1) min(-1) for C136A, and k(2) approximately 242 M(-1) min(-1) for C138A), although the yield of cluster transfer was found to be lower as a consequence of the enhanced lability of clusters in the derivative proteins. Minor variations in rate constant for the ISA Cys derivatives do not reflect any change in the affinity of binding to the apo Fd since k(2) was found to be independent of the concentration of apo Fd over the range of 1-25 microM. The pH dependence of cluster transfer rates was found to be similar for native and C136A ISA, with an observed pK(a) of 7.8 determined from the pH profiles for cluster transfer activity of each protein. The temperature dependence of the rate constant defining the cluster transfer reaction for the wild type versus this C136A ISA derivative is distinct (DeltaH* approximately 6.3 kcal mol(-1) and DeltaS* approximately -27.3 cal K(-1) mol(-1) for native and DeltaH* approximately 2.7 kcal mol(-1) and DeltaS* approximately -38.9 cal K(-1) mol(-1) for C136A ISA). Instability of the protein-bound cluster precluded a comparison with data from pH and temperature dependencies for the two other Cys derivatives. Experiments to determine the dependence of reaction rate constants on viscosity indicate cluster transfer is rate-limiting. A comparison of cross-species rate constants for cluster transfer to apo Fd targets from Homo sapiens and S. pombe demonstrated that the identity of the Fd is less critical for promoting cluster transfer from Sp ISA (at 25 degrees C, k(2) approximately 170 M(-1) min(-1) for Sp Fd and k(2) approximately 169 M(-1) min(-1) for Hs Fd). This contrasts with an earlier observation for ISU-mediated cluster assembly [Wu, S., et al. (2002) Biochemistry 41, 8876-8885], where the rates differed for Hs and Sp target Fd's, suggesting distinct binding sites for binding of holo ISA and ISU to apo Fd.     

NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein

NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and M?ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.     

Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide

Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-l-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA(*)). In the LS reaction, the 5'-dA(*) is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d(15) H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X(3)-C-X(2)-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X(4)-C-X(5)-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 +/- 0.5 irons and 6.4 +/- 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 +/- 0.1 irons and 3.6 +/- 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 +/- 0.1 irons and 4.7 +/- 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. M?ssbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S](2+). When wild-type LS was reconstituted with (57)Fe and sodium sulfide, it harbored considerably more iron (13.8 +/- 0.6) and sulfide (13.1 +/- 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. M?ssbauer spectroscopy of this protein revealed that only approximately 67% +/- 6% of the iron is in the form of [4Fe-4S](2+) clusters, amounting to 9.2 +/- 0.4 irons and 8.8 +/- 0.1 sulfides or 2 [4Fe-4S](2+) clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the M?ssbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.     

Identification of the binding region of the [2Fe-2S] ferredoxin in stearoyl-acyl carrier protein desaturase: insight into the catalytic complex and mechanism of action

Stearoyl-acyl carrier protein desaturase (Delta9D) catalyzes the O(2) and 2e(-) dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e(-) are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Delta9D complex by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies. The treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Delta9D contribute to cross-linking. The single substitutions of K60A, K56A, and K230A on Delta9D decreased the k(cat)/K(M) for Fd by 4-, 22-, and 2400-fold, respectively, as compared to wt Delta9D and a K41A substitution. The double substitution K56A/K60A decreased the k(cat)/K(M) for Fd by 250-fold, whereas the triple mutation K56A/K60A/K230A decreased the k(cat)/K(M) for Fd by at least 700 000-fold. These results strongly implicate the triad of K56, K60, and K230 of Delta9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and the carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd closer to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix 7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Delta9D and other protein-protein complexes.     

Unusual features in EPR and M?ssbauer spectra of the 2[4Fe-4Se]+ ferredoxin from Clostridium pasteurianum

The electronic and magnetic properties of the selenium-substituted 2[4Fe-4Se]2+/+ ferredoxin (Fd) from Clostridium pasteurianum have been investigated by EPR and M?ssbauer spectroscopy. The [4Fe-4Se]2+ clusters of oxidized Fd are diamagnetic and the M?ssbauer spectra are nearly identical to those of oxidized 2[4Fe-4S]2+ Fd. The addition of 2e- per molecule of Se-substituted Fd causes the simultaneous appearance of three EPR signals: one (g1,2,3 = 2.103, 1.940, 1.888) is reminiscent of [4Fe-4S]+ EPR spectra and accounts for 0.7 to 0.8 spin/molecule. The two others consist of a broad signal with g = 4.5, 3.5, and approximately 2 (0.7 to 0.8 spin/molecule) and of a narrow peak at g = 5.172 which is observed up to 60 K. Peculiar features are also present in the M?ssbauer spectra of 2[4Fe-4Se]+ Fd below 20 K: a subcomponent with lines near to +/- 4 mm/s and accounting for 20% of the total iron corresponds to two antiferromagnetically coupled sites in approximately a 3:1 ratio and displays fully developed paramagnetic hyperfine interactions at 4.2 K without any applied field. At 77 K, however, the reduced Se-substituted Fd yields a M?ssbauer spectrum similar to that of 2[4Fe-4S]+ Fd. The new EPR and M?ssbauer spectroscopic features of the 2[4Fe-4Se]+ Fd are attributed to S = 3/2 and S = 7/2 spin states which accompany the classical S = 1/2 state of [4Fe-4X]+ (X = S, Se) structures.     

alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is +153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) < -250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.     

M?ssbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase.

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. M?ssbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. M?ssbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.     

M?ssbauer and electron paramagnetic resonance studies of the cytochrome bf complex.

The (57)Fe-enriched cytochrome bf complex has been isolated from hydrocultures of spinach. It has been studied at different redox states by optical, EPR, and M?ssbauer spectroscopy. The M?ssbauer spectrum of the native complex at 190 K with all iron centers in the oxidized state reveals the presence of four different iron sites: low-spin ferric iron in cytochrome b [with an isomer shift (delta) of 0.20 mm/s, a quadrupole splitting (DeltaE(Q)) of 1.77 mm/s, and a relative area of 40%], low-spin ferric iron of cytochrome f (delta = 0.26 mm/s, DeltaE(Q) = 1.90 mm/s, and a relative area of 20%), and two high-spin ferric iron sites of the Rieske iron-sulfur protein (ISP) with a bis-cysteine and a bis-histidine ligated iron (delta(1) = 0.15 mm/s, DeltaE(Q1) = 0.70 mm/s, and a relative area of 20%, and delta(2) = 0.25 mm/s, DeltaE(Q2) = 0.90 mm/s, and a relative area of 20%, respectively). EPR and magnetic M?ssbauer measurements at low temperatures corroborate these results. A crystal-field analysis of the EPR data and of the magnetic M?ssbauer data yields estimates for the g-tensors (g(z)(), g(y)(), and g(x)()) of cytochrome b (3.60, 1.35, and 1.1) and of cytochrome f (3.51, 1.69, and 0.9). Addition of ascorbate reduces not only the iron of cytochrome f to the ferrous low-spin state (delta = 0.43 mm/s, DeltaE(Q) = 1.12 mm/s at 4.2 K) but also the bis-histidine coordinated iron of the Rieske 2Fe-2S center to the ferrous high-spin state (delta(2) = 0.73 mm/s, DeltaE(Q2) = -2.95 mm/s at 4.2 K). At this redox step, the M?ssbauer parameters of cytochrome b have not changed, indicating that the redox changes of cytochrome f and the Rieske protein did not change the first ligand sphere of the low-spin ferric iron in cytochrome b. Reduction with dithionite further reduces the two hemes of cytochrome b to the ferrous low-spin state (delta = 0.49 mm/s, DeltaE(Q) = 1.08 mm/s at 4.2 K). The spin Hamiltonian analysis of the magnetic M?ssbauer spectra at 4.2 K yields hyperfine parameters of the reduced Rieske 2Fe-2S center in the cytochrome bf complex which are very similar to those reported for the Rieske center from Thermus thermophilus [Fee, J. A., Findling, K. L., Yoshida, T., et al. (1984) J. Biol. Chem. 259, 124-133].     

Iron-sulfur center of biotin synthase and lipoate synthase

Biotin synthase and lipoate synthase are homodimers that are required for the C-S bond formation at nonactivated carbon in the biosynthesis of biotin and lipoic acid, respectively. Aerobically isolated monomers were previously shown to contain a (2Fe-2S) cluster, however, after incubation with dithionite one (4Fe-4S) cluster per dimer was obtained, suggesting that two (2Fe-2S) clusters had combined at the interface of the subunits to form the (4Fe-4S) cluster. Here we report M?ssbauer studies of (57)Fe-reconstituted biotin synthase showing that anaerobically prepared enzyme can accommodate two (4Fe-4S) clusters per dimer. The (4Fe-4S) cluster is quantitatively converted into a (2Fe-2S)(2+) cluster upon exposure to air. Reduction of the air-exposed enzyme with dithionite or photoreduced deazaflavin yields again (4Fe-4S) clusters. The (4Fe-4S) cluster is stable in both the 2+ and 1+ oxidation states. The M?ssbauer and EPR parameters were DeltaE(q) = 1.13 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and DeltaE(q) = 0.51 mm/s, delta = 0.85 mm/s, g(par) = 2.035, and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+). Considering that we find two (4Fe-4S) clusters per dimer, our studies argue against the early proposal that the enzyme contains one (4Fe-4S) cluster bridging the two subunits. Our study of lipoate synthase gave results similar to those obtained for BS: under strict anaerobiosis, lipoate synthase can accommodate a (4Fe-4S) cluster per subunit [DeltaE(q) = 1.20 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and g(par) = 2.039 and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+)], which reacts with oxygen to generate a (2Fe-2S)(2+) center.     

Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex

Rapid reaction studies presented herein show that ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) catalyzes electron transfer from spinach ferredoxin (Fd) to NADP+ via a ternary complex, Fd X FNR X NADP+. In the absence of NADP+, reduction of ferredoxin:NADP+ reductase by Fd was much slower than the catalytic rate: 37-80 s-1 versus at least 445 e-s-1; dissociation of oxidized spinach ferredoxin (Fdox) from one-electron reduced ferredoxin:NADP+ reductase (FNRsq) limited the reduction of FNR. This confirms the steady-state kinetic analysis of Masaki et al. (Masaki, R., Yoshikaya, S., and Matsubara, H. (1982) Biochim. Biophys. Acta 700, 101-109). Occupation of the NADP+ binding site of FNR by NADP+ or by 2',5'-ADP (a nonreducible NADP+ analogue) greatly increased the rate of electron transfer from Fd to FNR, releiving inhibition by Fdox. NADP+ (and 2',5'-ADP) probably facilitate the dissociation of Fdox; equilibrium studies have shown that nucleotide binding decreases the association of Fd with FNR (Batie, C. J. (1983) Ph.D. dissertation, Duke University; Batie, C. J., and Kamin, H. (1982) in Flavins and Flavoproteins VII (Massey, V., and Williams, C. H., Jr., eds) pp. 679-683, Elsevier, New York; Batie, C.J., and Kamin, H. (1982) Fed. Proc. 41, 888; and Batie, C.J., and Kamin, H. (1984) J. Biol. Chem. 259, 8832-8839). Premixing Fd with FNR was found to inhibit the reaction of the flavoprotein with NADP+ and with NADPH; thus, substrate binding may be ordered, NADP+ first, then Fd. FNRred and NADP+ very rapidly formed an FNRred X NADP+ complex with flavin to nicotinamide charge transfer bands. The Fdred X NADP+ complex then relaxed to an equilibrium species; the spectrum indicated a predominance of FNRox X NADPH charge-transfer complex. However, charge-transfer species were not observed during turnover; thus, their participation in catalysis of electron transfer from Fd to NADP+ remains uncertain. The catalytic rate of Fd to NADP+ electron transfer, as well as the rates of electron transfer from Fd to FNR, and from FNR to NADP+ were decreased when the reactants were in D2O; diaphorase activity was unaffected by solvent. On the basis of the data presented, a scheme for the catalytic mechanism of catalysis by FNR is presented.     

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.     

Mechanism of proton transfer in the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of (D)V and (D(2)O)V for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, (2)H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys(159) changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on V(max) and V/K(androsterone) and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on V(max) is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Br?nsted relationship between the log(V/K(d)(-base)Et) of the external amine (corrected for molecular size effects) and pK(a) is linear for the K159A mutant-catalyzed reaction at pH 10.4 (beta = 0.85 +/- 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr(155) to Lys(159) via 2'-OH of ribose.     

Site-directed mutagenesis of the PsaC subunit of photosystem I. F(b) is the cluster interacting with soluble ferredoxin.

The two [4Fe-4S] clusters F(A) and F(B) are the terminal electron acceptors of photosystem I (PSI) that are bound by the stromal subunit PsaC. Soluble ferredoxin (Fd) binds to PSI via electrostatic interactions and is reduced by the outermost iron-sulfur cluster of PsaC. We have generated six site-directed mutants of the green alga Chlamydomonas reinhardtii in which residues located close to the iron-sulfur clusters of PsaC are changed. The acidic residues Asp(9) and Glu(46), which are located one residue upstream of the first cysteine liganding cluster F(B) and F(A), respectively, were changed to a neutral or a basic amino acid. Although Fd reduction is not affected by the E46Q and E46K mutations, a slight increase of Fd affinity (from 1.3- to 2-fold) was observed by flash absorption spectroscopy for the D9N and D9K mutant PSI complexes. In the FA(2) triple mutant (V49I/K52T/R53Q), modification of residues located next to the F(A) cluster leads to partial destabilization of the PSI complex. The electron paramagnetic resonance properties of cluster F(A) are affected, and a 3-fold decrease of Fd affinity is observed. The introduction of positively charged residues close to the F(B) cluster in the FB(1) triple mutant (I12V/T15K/Q16R) results in a 60-fold increase of Fd affinity as measured by flash absorption spectroscopy and a larger amount of PsaC-Fd cross-linking product. The first-order kinetics are similar to wild type kinetics (two phases with t((1)/(2)) of <1 and approximately 4.5 microseconds) for all mutants except FB(1), where Fd reduction is almost monophasic with t((1)/(2)) < 1 microseconds. These data indicate that F(B) is the cluster interacting with Fd and therefore the outermost iron-sulfur cluster of PSI.     

Hyperproduction of recombinant ferredoxins in escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hs cA-fdx-ORF3 gene cluster.

Fe-S proteins acquire Fe-S clusters by an unknown post-translational mechanism. To study the in vivo synthesis of the Fe-S clusters, we constructed an experimental system to monitor the expressed ferredoxin (Fd) as a reporter of protein-bound Fe-S clusters assembled in Escherichia coli. Overexpression of five Fds in a T7 polymerase-based system led to the formation of soluble apoFds and mature holoFds, indicating that assembly of the Fe-S cluster into apoFd polypeptides is a rate-limiting step. We examined the coexpression of the E. coli ORF1-ORF2-iscS-iscU-iscA-hscB-hsc A-fdx-ORF3 gene cluster, which has recently been suggested to be involved in the formation or repair of Fe-S protein [Zheng, L., Cash, V.L., Flint, D.H., and Dean, D.R. (1998) J. Biol. Chem. 273, 13264-13272], with reporter Fds using compatible plasmids. The production of all five reporter holoFds examined was dramatically increased by the coexpression of the gene cluster, and apparent specificity to the polypeptides or to the type of Fe-S clusters was not observed. The increase in holoFd production was observed under the coexpression conditions in all culture media examined, with either 2 x YT medium or Terrific broth, and with or without supplemental cysteine or iron. These results indicate that the proteins encoded by the gene cluster are involved in the assembly of the Fe-S clusters in a wide variety of Fe-S proteins.     

The structure and computational analysis of Mycobacterium tuberculosis protein CitE suggest a novel enzymatic function

Fatty acid biosynthesis is essential for the survival of Mycobacterium tuberculosis and acetyl-coenzyme A (acetyl-CoA) is an essential precursor in this pathway. We have determined the 3-D crystal structure of M. tuberculosis citrate lyase beta-subunit (CitE), which as annotated should cleave protein bound citryl-CoA to oxaloacetate and a protein-bound CoA derivative. The CitE structure has the (beta/alpha)(8) TIM barrel fold with an additional alpha-helix, and is trimeric. We have determined the ternary complex bound with oxaloacetate and magnesium, revealing some of the conserved residues involved in catalysis. While the bacterial citrate lyase is a complex with three subunits, the M. tuberculosis genome does not contain the alpha and gamma subunits of this complex, implying that M. tuberculosis CitE acts differently from other bacterial CitE proteins. The analysis of gene clusters containing the CitE protein from 168 fully sequenced organisms has led us to identify a grouping of functionally related genes preserved in M. tuberculosis, Rattus norvegicus, Homo sapiens, and Mus musculus. We propose a novel enzymatic function for M. tuberculosis CitE in fatty acid biosynthesis that is analogous to bacterial citrate lyase but producing acetyl-CoA rather than a protein-bound CoA derivative.     

Functional studies of threonine 310 mutations in Glut1: T310I is pathogenic, causing Glut1 deficiency

We have previously reported on a patient with the Glut1 deficiency syndrome (Online Mendelian Inheritance in Man number 606777) carrying a heterozygous T310I missense mutation in the GLUT1 gene (Klepper, J., Wang, D., Fischbarg, J., Vera, J. C., Jarjour, I. T., O'Driscoll, K. R., and De Vivo, D. C. (1999) Neurochem. Res. 24, 587-594). To investigate the molecular basis for the associated functional deficit, we constructed T310A, T310S, and T310I human GLUT1 mutants for expression in Xenopus laevis oocytes via cRNA injection. For all mutants, glucose transport was decreased, and osmotic water permeability (Pf) was increased. Km values for 3-O-methylglucose (3-OMG) uptake under zero-trans influx and equilibrium exchange influx conditions were, respectively, 13 +/- 1 and 68 +/- 5 mm for wild-type Glut1, 5 +/- 1 and 25 +/- 6 mm for T310A, 6 +/- 3 and 30 +/- 6 mm for T310I, and 5 +/- 1 and 48 +/- 5 mm for T310S. Compared with wild-type Glut1, we determined the following. (a). Zero-trans and equilibrium exchange influx values of 3-OMG were significantly decreased, respectively, 15 and 5% in T310A, 8 and 3% in T310I, and 40 and 34% in T310S mutants. (b). Zero-trans efflux of 3-OMG and dehydroascorbic acid uptake were significantly decreased in mutants. (c). The relative Pf values for T310A, T310I, and T310S were increased 3-, 4.8-, and 3.5-fold compared with wild-type values. We found a very high negative correlation between the rate of glucose uptake and Pf (-0.93), and between hydropathy and uptake (-0.92), a moderate correlation between hydropathy and Pf (0.73), and a minimal correlation between uptake, Pf, and molecular weight. These findings are consistent with a central role for hydropathy rather than size at position 310 of this mutation.     

Purification of cytochrome P450 and ferredoxin, involved in bisphenol A degradation, from Sphingomonas sp. strain AO1

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450bisd), ferredoxin (Fd(bisd)), and ferredoxin reductase (Red(bisd)). We demonstrated that P450bisd and Fd(bisd) are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd(bisd) revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450(bisd), in the presence of Fd(bisd), Red(bisd), and NADH, was able to convert BPA. The K(m) and kcat values for BPA degradation were 85 +/- 4.7 microM and 3.9 +/- 0.04 min(-1), respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450bisd might exhibit strict specificity. Two BPA degradation products of the P450(bisd) system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.     

Activation of the M2 ion channel of influenza virus: a role for the transmembrane domain histidine residue.

To test the hypothesis that transmembrane domain histidine residue 37 of the M2 ion channel of influenza A virus mediates the low pH-induced activation of the channel, the residue was changed to glycine, glutamate, arginine, or lysine. The wild-type and altered M2 proteins were expressed in oocytes of Xenopus laevis and membrane currents were recorded. The mass of protein expressed in individual oocytes was measured using quantitative immunoblotting and correlated with membrane currents. Oocytes expressing the M2-H37G protein had a voltage-independent conductance with current-voltage relationship similar to that of the wild-type M2 channel. The conductance of the M2-H37G protein was reversibly inhibited by the M2 ion channel blocker amantadine and was only very slightly modulated by changes in pHout over the range pH 5.4 to pH 8.2. Oocytes expressing the M2-H37E protein also had a voltage-independent conductance with a current-voltage relationship similar to that of the wild-type M2 channel. The conductance of the M2-H37E protein was reversibly inhibited by amantadine and was also only very slightly modulated by changes in pHout over the range pH 5.4 to pH 8.2. These slight alterations in conductance of the mutant ion channels on changes in pHout are in striking contrast to the 50-fold change in conductance seen for the wild-type M2 channel over the range pH 4.5 to pH 8.2. The specific activity of the M2-H37G protein was 1.36 +/- 0.37 microA/ng and the specific activity of the M2-H37E protein was 30 +/- 3 microA/ng at pH 6.2.(ABSTRACT TRUNCATED AT 250 WORDS)