Br-rich tips of calcified crab claws are less hard but more fracture resistant: a comparison of mineralized and heavy-element biological materials.

We find that the spoon-like tips of the chelipeds (large claws) of the crab Pachygrapsus crassipes differ from the rest of the claw in that they are not calcified, but instead contain about 1% bromine--thus they represent a new example of a class of structural biological materials that contain heavy elements such as Zn, Mn, Fe, Cu, and Br bound in an organic matrix. X-ray absorption spectroscopy data suggest that the bromine is bound to phenyl rings, possibly in tyrosine. We measure a broad array of mechanical properties of a heavy-element biological material for the first time (abrasion resistance, coefficient of kinetic friction, energy of fracture, hardness, modulus of elasticity and dynamic mechanical properties), and we make a direct comparison with a mineralized tissue. Our results suggest that the greatest advantage of bromine-rich cuticle over calcified cuticle is resistance to fracture (the energy of fracture is about an order of magnitude greater than for calcified cuticle). The greatest advantage relative to unenriched cuticle, represented by ant mandible cuticle, is a factor of about 1.5 greater hardness and modulus of elasticity.The spoon-like tips gain additional fracture resistance from the orientation of the constituent laminae and from the viscoelasticity of the material. We suggest that fracture resistance is of greater importance in smaller organisms, and we speculate that one function of heavy elements in structural biological materials is to reduce molecular resonant frequencies and thereby increase absorption of energy from impacts.

The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a [4Fe-4S] cluster.

In type E succinate:quinone reductase (SQR), subunit SdhE (formerly SdhC) is thought to function as monotopic membrane anchor of the enzyme. SdhE contains two copies of a cysteine-rich sequence motif (CX(n)CCGX(m)CXXC), designated as the CCG domain in the Pfam database and conserved in many proteins. On the basis of the spectroscopic characterization of heterologously produced SdhE from Sulfolobus tokodaii, the protein was proposed in a previous study to contain a labile [2Fe-2S] cluster ligated by cysteine residues of the CCG domains. Using UV/vis, electron paramagnetic resonance (EPR), (57)Fe electron-nuclear double resonance (ENDOR) and Mössbauer spectroscopies, we show that after an in vitro cluster reconstitution, SdhE from S. solfataricus P2 contains a [4Fe-4S] cluster in reduced (2+) and oxidized (3+) states. The reduced form of the [4Fe-4S](2+) cluster is diamagnetic. The individual iron sites of the reduced cluster are noticeably heterogeneous and show partial valence localization, which is particularly strong for one unique ferrous site. In contrast, the paramagnetic form of the cluster exhibits a characteristic rhombic EPR signal with g (zyx) = 2.015, 2.008, and 1.947. This EPR signal is reminiscent of a signal observed previously in intact SQR from S. tokodaii with g (zyx) = 2.016, 2.00, and 1.957. In addition, zinc K-edge X-ray absorption spectroscopy indicated the presence of an isolated zinc site with an S(3)(O/N)(1) coordination in reconstituted SdhE. Since cysteine residues in SdhE are restricted to the two CCG domains, we conclude that these domains provide the ligands to both the iron-sulfur cluster and the zinc site.

Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA.

In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase.

Direct interaction of coenzyme M with the active-site Fe-S cluster of heterodisulfide reductase.

Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S=1/2 [4Fe-4S]3) cluster, the EPR spectrum of which is broadened in the presence of CoM-33SH [Duin, E.C., Madadi-Kahkesh, S., Hedderich, R., Clay, M.D. and Johnson, M.K. (2002) Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. FEBS Lett. 512, 263-268; Duin, E.C., Bauer, C., Jaun, B. and Hedderich, R. (2003) Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme. FEBS Lett. 538, 81-84]. These results provide indirect evidence that the disulfide binds to the iron-sulfur cluster during reduction. We report here direct structural evidence for this interaction from Se X-ray absorption spectroscopic investigation of HDR treated with the selenium analog of coenzyme M (CoM-SeH). Se K edge extended X-ray absorption fine structure confirms a direct interaction of the Se in CoM-SeH-treated HDR with an Fe atom of the Fe-S cluster at an Fe-Se distance of 2.4A.

Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme.

The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.

Structural characterization of Zn(II)-, Co(II)-, and Mn(II)-loaded forms of the argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli.

The Zn, Co, and Mn K-edge extended X-ray absorption fine structure (EXAFS) spectra of the N-acetyl-l-ornithine deacetylase (ArgE) from Escherichia coli, loaded with one or two equivalents of divalent metal ions (i.e., [Zn(II)_(ArgE)], [Zn(II)Zn(II)(ArgE)], [Co(II)_(ArgE)], [Co(II)Co(II)(ArgE)], [Mn(II)_(ArgE)], and [Mn(II)Mn(II)(ArgE)]), were recorded. The Fourier transformed data (FT) for [Zn(II)_(ArgE)], [Zn(II)Zn(II)(ArgE)], [Co(II)_(ArgE)] and [Co(II)Co(II)(ArgE)] are dominated by a peak at 2.05Å, that can be fit assuming five or six light atom (N,O) scatterers. Inclusion of multiple-scattering contributions from the outer-shell atoms of a histidine-imidazole ring resulted in reasonable Debye-Waller factors for these contributions and a slight reduction in the goodness-of-fit value (f'). Furthermore, the data best fit a model that included a M-M vector at 3.3 and 3.4Å for Zn(II) and Co(II), respectively, suggesting the formation of a dinuclear site. Multiple scattering contributions from the outer-shell atoms of a histidine-imidazole rings are observed at ~3 and 4Å for Zn(II)- and Co(II)-loaded ArgE suggesting at least one histidine ligand at each metal binding site. Likewise, EXAFS data for Mn(II)-loaded ArgE are dominated by a peak at 2.19Å that was best fit assuming six light atom (N,O) scatterers. Due to poor signal to noise ratios for the Mn EXAFS spectra, no Mn-Mn vector could be modeled. Peak intensities for [M(II)_(ArgE)] vs. [M(II)M(II)(ArgE)] suggest the Zn(II), Co(II), and Mn(II) bind to ArgE in a cooperative manner. Since no structural data has been reported for any ArgE enzyme, the EXAFS data reported herein represent the first structural glimpse for ArgE enzymes. These data also provide a structural foundation for the future design of small molecules that function as inhibitors of ArgE and may potentially function as a new class of antibiotics.

Characterization of the human smooth muscle cell secretome for regenerative medicine.

Smooth muscle cells (SMC) play a central role in maintaining the structural and functional integrity of muscle tissue. Little is known about the early in vitro events that guide the assembly of 'bioartificial tissue' (constructs) and recapitulate the key aspects of smooth muscle differentiation and development before surgical implantation. Biomimetic approaches have been proposed that enable the identification of in vitro processes which allow standardized manufacturing, thus improving both product quality and the consistency of patient outcomes. One essential element of this approach is the description of the SMC secretome, that is, the soluble and deposited factors produced within the three-dimensional (3D) extracellular matrix (ECM) microenvironment. In this study, we utilized autologous SMC from multiple tissue types that were expanded ex vivo and generated with a rigorous focus on operational phenotype and genetic stability. The objective of this study was to characterize the spatiotemporal dynamics of the first week of organoid maturation using a well-defined in vitro-like, 3D-engineered scale model of our validated manufacturing process. Functional proteomics was used to identify the topological properties of the networks of interacting proteins that were derived from the SMC secretome, revealing overlapping central nodes related to SMC differentiation and proliferation, actin cytoskeleton regulation, and balanced ECM accumulation. The critical functions defined by the Ingenuity Pathway Analysis included cell signaling, cellular movement and proliferation, and cellular and organismal development. The results confirm the phenotypic and functional similarity of the SMC generated by our platform technology at the molecular level. Furthermore, these data validate the biomimetic approaches that have been established to maintain manufacturing consistency.

Increased urothelial cell detection in the primary bladder smooth muscle cell cultures with dual MACS/qRT-PCR approach.

Bladder tissue has been regenerated in humans with neurogenic bladder using an implant produced from autologous urothelial (UC) and smooth muscle cells (SMC) expanded from bladder biopsies seeded onto a biodegradable synthetic scaffold. As the majority of bladder cancers are urothelial carcinomas (aka, transitional cell carcinoma), this 2-cell type autologous sourcing strategy presents significant challenges to product development. Entire bladders have been regenerated in cystectomized animals using a single-cell-type sourcing strategy: implants were seeded with bladder-derived SMC-only. Applying the bladder SMC-only sourcing strategy to produce clinical implants for bladder replacement or urinary diversion in bladder cancer patients requires methods for screening SMC cultures for the presence of potentially cancerous UC cells to provide evidence of SMC culture purity before seeding the scaffold. In this report, we show a 10-fold to 100-fold improvement in the sensitivity of qualitative and quantitative reverse-transcription PCR (qRT-PCR)-based assays for detecting UC positive for Cytokeratin 5 (CK5) in mixed SMC/UC cultures when the cell population was first subjected to magnetic activated cell sorting to enrich for cells expressing the epithelial cell adhesion molecule (known as EPCAM or CD326), a marker known to be present in normal UC and upregulated in the cancerous UC.

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.

Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity.

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn(2+) to modulate the assembly kinetics and morphology of congeners of the amyloid beta peptide (Abeta) associated with Alzheimer's disease. We now reveal a correlation among Abeta-Cu(2+) coordination, peptide self-assembly, and neuronal viability. By using the central segment of Abeta, HHQKLVFFA or Abeta(13-21), which contains residues H13 and H14 implicated in Abeta-metal ion binding, we show that Cu(2+) forms complexes with Abeta(13-21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-Abeta(13-21)H14A, alters metal coordination, allowing Cu(2+) to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Abeta can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Abeta self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

Modulating amyloid self-assembly and fibril morphology with Zn(II).

Metal ions (Zn(II)) are demonstrated as probes of amyloid structure in simple segments of the Abeta peptide, Abeta(13-21). By restricting the possible metal binding sites to His13/His14 dyad, we show that Zn2+ can specifically control the rate of self-assembly and dramatically regulate amyloid morphology via distinct coordination environments as characterized by X-ray absorption spectroscopy. The data establish that the single His13 is sufficient to coordinate Zn2+ productively for typical amyloid fiber formation, while a distinct Zn2+ coordination environment can be accessed in the presence of His13/Hi14 dyad to stabilize sheet/sheet associations and the transition to a ribbon/tube morphology.

Bottlenecks and roadblocks in high-throughput XAS for structural genomics.

Structural and functional characterization of the entire protein complement (the proteome) of an organism can provide an infrastructure upon which questions about biological pathways and systems biology can be framed. The technology necessary to perform this proteome-level structural and functional characterization is under development in numerous structural genomics and functional genomics initiatives. Given the ubiquity of metal active sites in a proteome, it seems appropriate to ask whether comprehensive local structural characterization of metal sites within a proteome (metalloproteomics) is either a valid or obtainable goal. With a proteome-wide knowledge of the active-site structures of all metalloproteins, one could start to ask how metal insertion, cluster assembly and metalloprotein expression are affected by growth conditions or developmental status etc. High-throughput X-ray absorption spectroscopy (HTXAS) is being developed as a technology for investigating the metalloproteome. In creating a pipeline from genome to metalloproteome, several bottlenecks to high-throughput determination of metal-site structures must be overcome. For example, automation of arraying small samples for XAS examination must be invented, automation of rapid data collection of multiple low-volume low-concentration samples must be developed, automation of data reduction and analysis must be perfected. Discussed here are the promises and the pitfalls of HTXAS development, including the results of initial feasibility experiments.

Rational design of a mononuclear metal site into the archaeal Rieske-type protein scaffold.

Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site (Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.

X-ray absorption spectroscopic analysis of reductive [2Fe-2S] cluster degradation in hyperthermophilic archaeal succinate:caldariellaquinone oxidoreductase subunits.

The biological [2Fe-2S] clusters play important roles in electron transfer and cellular signaling for a variety of organisms from archaea, bacteria to eukarya. The two recombinant hyperthermophilic archaeal [2Fe-2S] cluster-binding proteins, SdhC and the N-terminal domain fragment of SdhB, of Sulfolobus tokodaii respiratory complex II overproduced in Escherichia coli are thermostable as isolated, but moderately sensitive to reduction with excess dithionite. We used iron K-edge X-ray absorption spectroscopy to monitor the structural changes of their Fe sites in the irreversible [2Fe-2S] cluster degradation process. Regardless of the differences in the cluster-ligating cysteine motifs and the XAS-detectable [2Fe-2S](2+) cluster environments, a complete reductive breakdown of the [2Fe-2S] clusters resulted in the appearance of a new Fourier transform (FT) peak at approximately 3.3 A with a concomitant loss of the Fe-Fe interaction at ca. 2.7 A for both proteins. On the basis of the unambiguous assignment of the 3.3 A FT peak, our results suggest that a biological [2Fe-2S] cluster breakdown under reducing conditions generally releases Fe(2+) from the polypeptide chain into the aqueous solution, and the Fe(2+) might then be recruited as a secondary ferrous iron source for de novo biosynthesis and/or regulation of iron-binding enzymes in the cellular system.

Structural elements of metal selectivity in metal sensor proteins.

Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR are homologous zinccobalt-responsive and nickelcobalt-responsive transcriptional repressors in vivo, respectively, and members of the ArsRSmtB superfamily of prokaryotic metal sensor proteins. We show here that Zn(II) is the most potent negative allosteric regulator of czr operatorpromoter binding in vitro with the trend Zn(II)>Co(II)Ni(II), whereas the opposite holds for the binding of NmtR to the nmt operatorpromoter, Ni(II)>Co(II)>Zn(II). Characterization of the metal coordination complexes of CzrA and NmtR by UVvisible and x-ray absorption spectroscopies reveals that metals that form four-coordinate tetrahedral complexes with CzrA [Zn(II) and Co(II)] are potent regulators of DNA binding, whereas metals that form five- or six-coordinate complexes with NmtR [Ni(II) and Co(II)] are the strongest allosteric regulators in this system. Strikingly, the Zn(II) coordination complexes of CzrA and NmtR cannot be distinguished from one another by x-ray absorption spectroscopy, with the best fit a His-3-carboxylate complex in both cases. Inspection of the primary structures of CzrA and NmtR, coupled with previous functional data, suggests that three conserved His and one Asp from the C-terminal alpha5 helix donate ligands to create a four-coordinate complex in both CzrA and NmtR, with NmtR uniquely capable of expanding its coordination number in the Ni(II) and Co(II) complexes by recruiting additional His ligands from a C-terminal extension of the alpha5 helix.

Spectroscopic determination of the thermodynamics of cobalt and zinc binding to GATA proteins.

Vertebrate GATA proteins regulate processes that are vital to development, and each possesses two tandem GATA finger domains: an N-terminal GATA finger and a C-terminal GATA finger. These GATA fingers require Zn(2+) to fold, to bind DNA recognition elements, and to regulate transcription. While the GATA-1 C-terminal finger is necessary and sufficient to bind to single GATA DNA sites, the N-terminal finger interacts with DNA such that the double finger unit (DF domain) has a binding and transactivation profile that is tuned by the DNA-binding site. Co(2+) was used as a spectroscopic probe in a series of competition titrations to determine the affinity of Co(2+) and Zn(2+) for the C-terminal finger from chicken GATA-1 and the double finger from human GATA-1 (referred to in this report as CF and DF). For CF, these experiments yielded K(b)(Co) = 1.0 (+/-1.3) x 10(7) M(-1) and K(b)(Zn) = 2.0 (+/-1.3) x 10(10) M(-1). For DF, these experiments yielded equilibrium constants for the process of two M(2+) binding to form M(2+)(2)-DF of beta(2)(Co) = 2.5 (+/-1.6) x 10(14) M(-2) and beta(2)(Zn) = 6.3 (+/-2.5) x 10(20) M(-2). The ZnS(4) coordination environment of Zn(2+)-bound CF was confirmed with X-ray absorption spectroscopy. A detailed analysis of these data suggests that the N-terminal and C-terminal fingers of DF act as independent and identical Zn(2+)-binding sites and each finger binds Zn(2+) with an affinity equivalent to that of CF.

A stable mercury-containing complex of the organomercurial lyase MerB: catalysis, product release, and direct transfer to MerA.

Bacteria isolated from organic mercury-contaminated sites have developed a system of two enzymes that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. Both enzymes are encoded on the mer operon and require sulfhydryl-bound substrates. The first enzyme is an organomercurial lyase (MerB), and the second enzyme is a mercuric ion reductase (MerA). MerB catalyzes the protonolysis of the carbon-mercury bond, resulting in the formation of a reduced carbon compound and inorganic ionic mercury. Of several mercury-containing MerB complexes that we attempted to prepare, the most stable was a complex consisting of the organomercurial lyase (MerB), a mercuric ion, and a molecule of the MerB inhibitor dithiothreitol (DTT). Nuclear magnetic resonance (NMR) spectroscopy and extended X-ray absorption fine structure spectroscopy of the MerB/Hg/DTT complex have shown that the ligands to the mercuric ion in the complex consist of both sulfurs from the DTT molecule and one cysteine ligand, C96, from the protein. The stability of the MerB/Hg/DTT complex, even in the presence of a large excess of competing cysteine, has been demonstrated by NMR and dialysis. We used an enzyme buffering test to determine that the MerB/Hg/DTT complex acts as a substrate for the mercuric reductase MerA. The observed MerA activity is higher than the expected activity assuming free diffusion of the mercuric ion from MerB to MerA. This suggests that the mercuric ion can be transferred between the two enzymes by a direct transfer mechanism.

Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus.

We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae is a dinuclear metallohydrolase.

The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 A, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 A, respectively. A second-shell feature at ca. 3.34 A appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear Zn(II) active site. Since no X-ray crystallographic data are available for any DapE enzyme, these data provide the first glimpse at the active site of DapE enzymes. In addition, the EXAFS data for DapE incubated with two competitive inhibitors, 2-carboxyethylphosphonic acid and 5-mercaptopentanoic acid, are also presented.