Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement.

Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here we report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure.     

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred over serine ligation.

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives three of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence at positions 39-45 while the fourth ligand, Cys20, is provided by a distal portion of the sequence. Previously we reported that the site-directed mutation of Cys20 to Ala (C20A protein) resulted in the formation of a new [4Fe-4S] cluster that obtained its fourth ligand from Cys24, a free cysteine in the native structure. That ligand exchange required significant protein rearrangement. Here we report the conversion of Cys20 to Ser (C20S protein), which gives the protein the opportunity either to retain the native structure and use the Ser20 O gamma as a ligand or to rearrange and use Cys24. X-ray crystallography demonstrates that the cluster does not use the Ser20 O gamma as a ligand; rather it rearranges to use Cys24. In the C20S protein the [4Fe-4S] cluster has altered stability and redox properties relative to either C20A or the native protein.     

Structure of ferredoxin I from Azotobacter vinelandii.

The structure of Azotobacter vinelandii ferredoxin I (Av FdI, 106 amino acids) has been redetermined, based on x-ray diffraction data from tetragonal crystals of the native protein and two heavy atom derivatives. The current model differs greatly from the one previously reported and is in agreement with arguments based on various spectroscopic and other methods. The unit cell parameters are a = b = 55.62 A and c = 95.51 A, whereas the space group was found to be P4(1)2(1)2 instead of P4(3)2(1)2. The sequence of the first half of Av FdI is closely homologous with ferredoxin from Peptococcus aerogenes (Pa Fd, 54 amino acids) and the fold of the corresponding chain is almost identical. The ligands of the 3Fe complex are Cys-8, -16, and -49, corresponding to three of the four ligands in complex I of Pa Fd; the ligands of the 4Fe complex are Cys-20, -39, -42, and -45, corresponding to the four ligands in complex II of Pa Fd.     

Spectroscopic studies of ferricyanide oxidation of Azotobacter vinelandii ferredoxin I.

The Fe(CN)3-(6) oxidation of the crystallographically characterized [[3Fe-3S], [4Fe-4S]] ferredoxin I of Azotobacter vinelandii has been studied using absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopies. A paramagnetic intermediate is observed en route to Fe-S cluster-free apoprotein, possessing an anisotropic g approximately equal to 2 EPR signal, surviving to temperatures greater than 77 K. This species is shown to result from 3-electron oxidation of the [4Fe-4S] cluster, without modification of the [3Fe-3S] cluster. However, it does not give rise to observable paramagnetic magnetic circular dichroism in the visible-near UV spectral region and is therefore neither an oxidized HIPIP [4Fe-4S] cluster nor an oxidized [3Fe-3S] cluster. We identify the paramagnetic species as a cysteinyldisulfide radical formed on dissociation of an oxidized cysteinate and an oxidized sulfide ion from the [4Fe-4S] cluster. This conclusion is consistent with the observed reaction stoichiometry, the spectroscopic results obtained, known EPR spectra of disulfide radicals, and the reconstitution of the native [4Fe-4S] cluster by dithiothreitol alone. This reaction, earlier interpreted as a HIPIP-type oxidation, is a previously uncharacterized oxidation reaction of [4Fe-4S] clusters.     

Iron-sulfur stoichiometry and structure of iron-sulfur clusters in three-iron proteins: Evidence for [3Fe-4S] clusters

Beef heart aconitase contains 3Fe clusters in its inactive and 4Fe clusters in its active form. The fully active form can be restored from the inactive one by insertion of Fe(2+), whereas S(2-) is not required. Chemical analyses for iron and labile sulfide yield Fe/S(2-) ratios of 0.66-0.74 for the inactive and 0.90-1.03 for the active form. Sulfane sulfur (S(0)) was not detected. We propose on the basis of these data that the inactive form may arise from the active one by loss of one iron only per cluster with the sulfur remaining as S(2-) in a [3Fe-4S] structure. Measurements by extended x-ray absorption fine structure (EXAFS) spectroscopy on the 3Fe form of aconitase yield a Fe..S distance of 2.24 A and a Fe..Fe distance of 2.71 A. This Fe..Fe distance is in agreement with that obtained by EXAFS on ferredoxin II of Desulfovibrio gigas, another 3Fe protein, but disagrees with Fe..Fe distances observed for the 3Fe cluster of Azotobacter vinelandii ferredoxin I by x-ray diffraction-namely, 4.1 A. We suggest that this difference may be due to the presence of a [3Fe-3S] structure in the Azotobacter ferredoxin I crystals vs. a [3Fe-4S] structure in liquid or frozen solutions of aconitase. The [3Fe-3S] cluster has been shown to have a relatively flat twist-boat structure, whereas a [3Fe-4S] cluster could be expected to essentially maintain the compact structure of the [4Fe-4S] cluster. This would explain the differences in Fe..Fe distances. Two possible structural models for a [3Fe-4S] cluster are discussed.     

Flavodoxin hydroquinone reduces Azotobacter vinelandii Fe protein to the all-ferrous redox state with a S = 0 spin state

Azotobacter vinelandii flavodoxin hydroquinone (FldHQ) is a physiological reductant to nitrogenase supporting catalysis that is twice as energy efficient (ATP/2e- = 2) as dithionite (ATP/2e- = 4). This catalytic efficiency results from reduction of Fe protein from A. vinelandii (Av2) to the all-ferrous oxidation state ([Fe4S4]0), in contrast to dithionite, which only reduces Av2 to the [Fe4S4]1+ state. Like FldHQ, Ti(III) citrate yields ATP/2e- = 2, and Ti(III)-reduced [Fe4S4]0 Av2 has a S = 4 spin state and characteristic Mossbauer spectrum, a parallel mode g = 16.4 EPR signal, and a shoulder at 520 nm in its UV-vis spectrum, each of which distinguish the S = 4 [Fe4S4]0 Av2 from other states. In this study, we demonstrate that FldHQ makes [Fe4S4]0 Av2, which is sufficiently characterized to demonstrate unique physical properties that distinguish it from the previously characterized Ti(III)-reduced [Fe4S4]0 Av2. In particular, Evans NMR magnetic susceptibility and EPR measurements indicate that FldHQ-reduced [Fe4S4]0 Av2 has an S = 0 spin state (like [Fe4S4]2+ Av2). There is no g = 16.4 EPR signal and no shoulder at 520 nm in its absorbance spectrum, which resembles that of [Fe4S4]1+ Av2. That the physiological reductant to Av2 is capable of forming [Fe4S4]0 Av2 has important implications for in vivo nitrogenase activity.     

Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli.

A cDNA fragment encoding human ferredoxin, a mitochondrial [2Fe-2S] protein, was introduced into Escherichia coli by using an expression vector based on the approach of Nagai and Th?gersen [Nagai, K. & Th?gersen, M. C. (1984) Nature (London) 309, 810-812]. Expression was under control of the lambda PL promoter and resulted in production of ferredoxin as a cleavable fusion protein with an amino-terminal fragment derived from bacteriophage lambda cII protein. The fusion protein was isolated from the soluble fraction of induced cells and was specifically cleaved to yield mature recombinant ferredoxin. The recombinant protein was shown to be identical in size to ferredoxin isolated from human placenta (13,546 Da) by NaDodSO4/PAGE and partial amino acid sequencing. E. coli cells expressing human ferredoxin were brown in color, and absorbance and electron paramagnetic resonance spectra of the purified recombinant protein established that the [2Fe-2S] center was assembled and incorporated into ferredoxin in vivo. Recombinant ferredoxin was active in steroid hydroxylations when reconstituted with cytochromes P-450scc and P-450(11) beta and exhibited rates comparable to those observed for ferredoxin isolated from human placenta. This expression system should be useful in production of native and structurally altered forms of human ferredoxin for studies of ferredoxin structure and function.     

Synthetic analogues of [Fe4S4(Cys)3(His)] in hydrogenases and [Fe4S4(Cys)4] in HiPIP derived from all-ferric [Fe4S4{N(SiMe3)2}4]

The all-ferric [Fe₄S₄]⁴⁺ cluster [Fe₄S₄{N(SiMe₃)₂}₄] 1 and its one-electron reduced form [1]^- serve as convenient precursors for the synthesis of 3∶1-site differentiated [Fe₄S₄] clusters and high-potential iron-sulfur protein (HiPIP) model clusters. The reaction of 1 with four equivalents (equiv) of the bulky thiol HSDmp (Dmp = 2,6-(mesityl)₂C₆H₃, mesityl = 2,4,6-Me₃C₆H₂) followed by treatment with tetrahydrofuran (THF) resulted in the isolation of [Fe₄S₄(SDmp)₃(THF)₃] 2. Cluster 2 contains an octahedral iron atom with three THF ligands, and its Fe(S)₃(O)₃ coordination environment is relevant to that in the active site of substrate-bound aconitase. An analogous reaction of [1]^- with four equiv of HSDmp gave [Fe₄S₄(SDmp)₄]^- 3, which models the oxidized form of HiPIP. The THF ligands in 2 can be replaced by tetramethyl-imidazole (Me₄Im) to give [Fe₄S₄(SDmp)₃(Me₄Im)] 4 modeling the [Fe₄S₄(Cys)₃(His)] cluster in hydrogenases, and its one-electron reduced form [4]^- was synthesized from the reaction of 3 with Me₄Im. The reversible redox couple between 3 and [3]^- was observed at E_1/2 = -820 mV vs. Ag/Ag⁺, and the corresponding reversible couple for 4 and [4]^- is positively shifted by +440 mV. The cyclic voltammogram of 3 also exhibited a reversible oxidation couple, which indicates generation of the all-ferric [Fe₄S₄]⁴⁺ cluster, [Fe₄S₄(SDmp)₄].     

A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of β-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the β-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron–sulfur cluster that is necessary for activity. These findings provide an example of an iron–sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)–dependent amide bond formation in a natural product biosynthetic pathway.

Amide bond formation in natural product biosynthesis can be catalyzed by nonribosomal peptide synthetases (NRPSs) and ATP-grasp ligases including the ATP-dependent activation of amino acid substrates. The carboxyl group is activated by phosphorylation and adenylation, respectively, in ATP-grasp ligase and NRPS catalyzed reactions (1). Recently, a new family of enzymes has been found to catalyze amide bond formation using aminoacyl-transfer RNA (aa-tRNA) as an activated cosubstrate. aa-tRNA plays a profound role in cells connecting the messenger RNA (mRNA) and protein synthesis at the ribosome in primary metabolism (2). Interestingly, recent studies have revealed that aa-tRNA can also be involved in natural product biosynthesis (3). aa-tRNA–dependent enzymes involved in natural product biosynthesis mainly form three groups: amide bond-forming ligases homologous with FemX peptidyltransferases from cell wall biosynthesis (4), synthase enzymes including the cyclodipeptide synthase family (5, 6), and dehydratase enzymes in RiPP (ribosomally synthesized and posttranslationally modified peptide) synthesis (7).Blasticidin S (BS), a representative of the amino hexose pyrimidine nucleoside antibiotics, was first isolated from Streptomyces griseochromogenes (8). The structure of BS features a C2′, C3′-dehydrated pyrose ring that is connected with cytosine at C1′ and β-arginine at C4′ (9). BS shows a broad spectrum of biological activities and had been widely used as a fungicide to protect rice from blast diseases (10, 11). Presently, BS is commonly used as an efficient selection antibiotic for transformed mammalian cells that express appropriate resistance genes (12, 13).The structural features and commercial value of BS stimulated interest in its biosynthetic studies. Early feeding experiments and characterization of related metabolites determined that glucose, cytosine, L-arginine, and methionine are the basic precursors for BS biosynthesis (14). The biosynthesis gene cluster was reported in 2003 and enabled a proposal for the BS biosynthetic pathway (Fig. 1) (15). However, difficulty carrying out gene knockout experiments in the native producer, S. griseochromogenes, hindered the further characterization of each biosynthetic step. More recently, the BS biosynthetic cluster was engrafted into the chromosome of Streptomyces lividans to generate the genetically stable mutant strain WJ2, which is able to produce BS and thereby facilitating in vivo studies of the function of BS biosynthetic genes (13). Gene deletions in WJ2 determined the essential genes for BS biosynthesis including blsD-blsL that are transcribed in one direction and the divergently transcribed blsM. The first committed step of BS biosynthesis is the hydrolysis of cytidine monophosphate by BlsM to produce free cytosine, which is then condensed with UDP-glucuronic acid by BlsD to form cytosyl-glucuronic acid (CGA) (15, 16). The process from CGA to cytosinine, a putative intermediate for BS biosynthesis, remains to be determined. A radical SAM protein BlsE and an aminotransferase BlsH are possibly involved in this transformation (13, 15). The β-arginine moiety of BS is derived from L-arginine through a radical-mediated reaction catalyzed by BlsG, a 2,3-arginine aminomutase (15, 17). It remains unknown whether BlsI, a putative ligase, is involved in the formation of DBS (demethylblasticidin S) by coupling of the carboxyl group of β-arginine and the amido group at C4′ of cytosinine since mutant WJ2∆blsI only accumulates the very early intermediate CGA. It is worth noting that DBS cannot be directly methylated to BS by the N-methyltransferase BlsL, which was confirmed by in vitro biochemical characterization (18).Open in a separate windowFig. 1.The proposed biosynthetic pathway of BS. CMP: Cytidine 5-monophosphate; AdoMet: S-Adenosylmethionine.Addition of leucine to DBS at the β-amino group of the arginine side chain forms leucyldemethylblasticidin S (LDBS) which is then methylated by BlsL to form the penultimate product leucylblasticidin S (LBS) (18). Finally, PepN catalyzes the maturation of BS biosynthesis via the hydrolysis of the leucyl group of LBS (19). The cell toxicity of LDBS and LBS are much lower than DBS and BS. Therefore, it is proposed that the circuitous modifications of BS and DBS with a leucyl group function as the self-resistance mechanism during BS biosynthesis (16). Attempts to form LDBS from DBS in a S. griseochromogenes cell-free system were not successful. A plausible reason for the failure of LDBS formation was that even if LDBS could be formed, it would be readily hydrolyzed back to DBS by the conserved aminopeptidase PepN in the cell-free system (16, 19).The intermediacy of LBS and LDBS hints at the necessity of an extra ligase other than BlsI in BS biosynthesis (15). In this report, we disclose the mechanism of the cryptic leucylation involved in BS biosynthesis. By combining in vivo gene inactivation and in vitro biochemical assays, we demonstrate that BlsK catalyzes the tRNA-dependent transfer of a leucyl group to the DBS β-arginine moiety by directing leucyl-tRNA^Leu to the BS biosynthetic pathway. More interestingly, BlsK is determined to be an iron–sulfur enzyme that does not show similarity with any known iron–sulfur enzymes. BlsK contains a [3Fe-4S] cluster that is critical for its activity. An iron–sulfur protein was shown to be involved in the amide bond formation. These results pave the way to fully understand the self-resistance and biosynthesis of BS.     

Azotobacter vinelandii RNA POLYMERASE, VII. ENZYME TRANSITIONS DURING UNPRIMED r[I-C] SYNTHESIS

Transitions in the state of RNA polymerase were demonstrated during the unprimed synthesis of the r[I-C] copolymer. No detectable change in the usual dimer-monomer pattern was noted during the lag phase (0-25 min at 37 degrees ) after analysis of the reaction mixture by acrylamide gel electrophoresis. At the end of the lag phase, a major alteration in the electrophoretic pattern occurred, marked by the disappearance of the dimer-monomer bands and the concomitant appearance of a series of monomer-r[I-C] copolymer complexes. As these complexes of r[I-C] copolymer with one or more polymerase monomer were formed, an enzymatically inactive component (gamma protein) of the polymerase was displaced. During the phase of rapid r[I-C] copolymer synthesis, the active form of the A. vinelandii RNA polymerase was the r[I-C] monomer lacking the gamma protein.     

Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal.

The structure of activated pig heart aconitase [citrate(isocitrate) hydro-lyase, EC 4.2.1.3] containing a [4Fe-4S] cluster has been refined at 2.5-A resolution to a crystallographic residual of 18.2%. Comparison of this structure to the recently determined 2.1-A resolution structure of the inactive enzyme containing a [3Fe-4S] cluster, by difference Fourier analysis, shows that upon activation iron is inserted into the structure isomorphously. The common atoms of the [3Fe-4S] and [4Fe-4S] cores agree within 0.1 A; the three common cysteinyl S gamma ligand atoms agree within 0.25 A. The fourth ligand of the Fe inserted into the [3Fe-4S] cluster is a water or hydroxyl from solvent, consistent with the absence of a free cysteine ligand in the enzyme active site cleft and the isomorphism of the two structures. A water molecule occupies a similar site in the crystal structure of the inactive enzyme.     

Crystal structure of 4-hydroxybutyryl-CoA dehydratase: radical catalysis involving a [4Fe-4S] cluster and flavin

Dehydratases catalyze the breakage of a carbon-oxygen bond leading to unsaturated products via the elimination of water. The 1.6-A resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the gamma-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of dehydratases with an unprecedented active site architecture. A [4Fe-4S](2+) cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide (FAD) moiety. The structure provides insight into the function of these ubiquitous prosthetic groups in the chemically nonfacile, radical-mediated dehydration of 4-hydroxybutyryl-CoA. The substrate can be bound between the [4Fe-4S](2+) cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversion. Our results raise interesting questions regarding the mechanism of acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, and address the divergent evolution of the ancestral common gene.     

NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein

The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+, +) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.     

A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans

In anaerobic microorganisms employing the acetyl-CoA pathway, acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) form a complex (ACS/CODH) that catalyzes the synthesis of acetyl-CoA from CO, a methyl group, and CoA. Previously, a [4Fe-4S] cubane bridged to a copper-nickel binuclear site (active site cluster A of the ACS component) was identified in the ACS(Mt)/CODH(Mt) from Moorella thermoacetica whereas another study revealed a nickel-nickel site in the open form of ACS(Mt), and a zink-nickel site in the closed form. The ACS(Ch) of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans was found to exist as an 82.2-kDa monomer as well as in a 1:1 molar complex with the 73.3-kDa CODHIII(Ch). Homogeneous ACS(Ch) and ACS(Ch)/CODHIII(Ch) catalyzed the exchange between [1-(14)C]acetyl-CoA and (12)CO with specific activities of 2.4 or 5.9 micromol of CO per min per mg, respectively, at 70 degrees C and pH 6.0. They also catalyzed the synthesis of acetyl-CoA from CO, methylcobalamin, corrinoid iron-sulfur protein, and CoA with specific activities of 0.14 or 0.91 micromol of acetyl-CoA formed per min per mg, respectively, at 70 degrees C and pH 7.3. The functional cluster A of ACS(Ch) contains a Ni-Ni-[4Fe-4S] site, in which the positions proximal and distal to the cubane are occupied by Ni ions. This result is apparent from a positive correlation of the Ni contents and negative correlations of the Cu or Zn contents with the acetyl-CoA/CO exchange activities of different preparations of monomeric ACS(Ch), a 2.2-A crystal structure of the dithionite-reduced monomer in an open conformation, and x-ray absorption spectroscopy.