Nathaniel J. Cosper

Structural elements of metal selectivity in metal sensor proteins

Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR are homologous zinc/cobalt-responsive and nickel/cobalt-responsive transcriptional repressors in vivo, respectively, and members of the ArsR/SmtB superfamily of prokaryotic metal sensor proteins. We show here that Zn(II) is the most potent negative allosteric regulator of czr operator/promoter binding in vitro with the trend Zn(II)>Co(II)≫Ni(II), whereas the opposite holds for the binding of NmtR to the nmt operator/promoter, Ni(II)>Co(II)>Zn(II). Characterization of the metal coordination complexes of CzrA and NmtR by UV/visible 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 α5 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 α5 helix.

Structural and kinetic characterization of an archaeal beta-class carbonic anhydrase.

The beta-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) beta-class carbonic anhydrase indicate that the secondary structure of the beta-class enzymes is predominantly alpha-helical, unlike that of the alpha- or gamma-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k(cat) and k(cat)/K(m) for CO(2) hydration are pH dependent. The steady-state parameter k(cat) is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k(cat) is 2.1-fold higher in H(2)O than in D(2)O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k(cat)/K(m) is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic beta-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic beta-class enzymes and firmly establish that the alpha-, beta-, and gamma-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.

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 (Em = -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 (Cys42 and Cys61) 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 His64 → Cys variant (H64C) than the His44 → 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 15N-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.

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.

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.

Coordination and geometry of the nickel atom in active methyl-coenzyme M reductase from Methanothermobacter marburgensis as detected by X-ray absorption spectroscopy

Abstract. Methyl-coenzyme M reductase (MCR) catalyzes the reduction of methyl-coenzyme M (CH3–S–CoM) to methane. The enzyme contains as a prosthetic group the nickel porphinoid F430 which in the active enzyme is in the EPR-detectable Ni(I) oxidation state. Crystal structures of several inactive Ni(II) forms of the enzyme but not of the active Ni(I) form have been reported. To obtain structural information on the active enzyme–substrate complex we have now acquired X-ray absorption spectra of active MCR in the presence of either CH3–S–CoM or the substrate analog coenzyme M (HS–CoM). For both MCR complexes the results are indicative of the presence of a five-coordinate Ni(I), the five ligands assigned as four nitrogen ligands from F430 and one oxygen ligand. Analysis of the spectra did not require the presence of a sulfur ligand indicating that CH3–S–CoM and HS–CoM were not coordinated via their sulfur atom to nickel in detectable amounts. As a control, X-ray absorption spectra were evaluated of three enzymatically inactive MCR forms, MCR-silent, MCR-ox1-silent and MCR-ox1, in which the nickel is known to be six-coordinate. Comparison of the edge position of the X-ray absorption spectra revealed that the Ni(I) in the active enzyme is more reduced than the Ni in the two EPR-silent Ni(II) states. Surprisingly, the edge position of the EPR-active MCR-ox1 state was found to be the same as that of the two silent states indicating similar electron density on the nickel. Electronic supplementary material is available if you access this article at http://www.dx.doi.org/10.1007/s00775-002-0399-2. On that page (frame on the left side), a link takes you directly to the supplementary material.

Redox-dependent structural changes in archaeal and bacterial Rieske-type [2Fe-2S] clusters.

Proteins containing Rieske‐type [2Fe‐2S] clusters play important roles in many biological electron transfer reactions. Typically, [2Fe‐2S] clusters are not directly involved in the catalytic transformation of substrate, but rather supply electrons to the active site. We report herein X‐ray absorption spectroscopic (XAS) data that directly demonstrate an average increase in the iron–histidine bond length of at least 0.1 Å upon reduction of two distantly related Rieske‐type clusters in archaeal Rieske ferredoxin from Sulfolobus solfataricus strain P‐1 and bacterial anthranilate dioxygenases from Acinetobacter sp. strain ADP1. This localized redox‐dependent structural change may fine tune the protein–protein interaction (in the case of ARF) or the interdomain interaction (in AntDO) to facilitate rapid electron transfer between a lower potential Rieske‐type cluster and its redox partners, thereby regulating overall oxygenase reactions in the cells.

Characterization of metal-substituted Klebsiella aerogenes urease

Abstract Urease possesses a dinuclear Ni active site with the protein providing a bridging carbamylated lysine residue as well as an aspartyl and four histidyl ligands. The apoprotein can be activated in vitro by incubation with bicarbonate/CO2 and Ni(II); however, only ∼15% forms active enzyme (Ni-CO2-ureaseA), with the remainder forming inactive carbamylated Ni-containing protein (Ni-CO2-ureaseB). In the absence of CO2, apoprotein plus Ni(II) forms a distinct inactive Ni-containing species (Ni-urease). The studies described here were carried out to better define the metal-binding sites for the inactive Ni-urease and Ni-CO2-ureaseB species, and to examine the properties of various forms of Co-, Mn-, and Cu-substituted ureases. X-ray absorption spectroscopy (XAS) indicated that the two Ni atoms present in the Ni-urease metallocenter are coordinated by an average of two histidines and 3–4 N/O ligands, consistent with binding to the usual enzyme ligands with the lysine carbamate replaced by solvent. Neither XAS nor electronic spectroscopy provided evidence for thiolate ligation in the inactive Ni-containing species. By contrast, comparative studies of Co-CO2-urease and its C319A variant by electronic spectroscopy were consistent with a portion of the two Co being coordinated by Cys319. Whereas the inactive Co-CO2-urease possesses a single histidyl ligand per metal, the species formed using C319A apoprotein more nearly resembles the native metallocenter and exhibits low levels of activity. Activity is also associated with one of two species of Mn-CO2-urease. A crystal structure of the inactive Mn-CO2-urease species shows a metallocenter very similar in structure to that of native urease, but with a disordering of the Asp360 ligand and movement in the Mn-coordinated solvent molecules. Cu(II) was bound to many sites on the protein in addition to the usual metallocenter, but most of the adventitious metal was removed by treatment with EDTA. Cu-treated urease was irreversibly inactivated, even in the C319A variant, and was not further characterized. Metal speciation between Ni, Co, and Mn most affected the higher of two pKa values for urease activity, consistent with this pKa being associated with the metal-bound hydrolytic water molecule. Our results highlight the importance of precisely positioned protein ligands and solvent structure for urease activity.

Spectroscopic investigation of selective cluster conversion of archaeal zinc-containing ferredoxin from Sulfolobus sp. strain 7.

Archaeal zinc-containing ferredoxin fromSulfolobus sp. strain 7 contains one [3Fe-4S] cluster (cluster I), one [4Fe-4S] cluster (cluster II), and one isolated zinc center. Oxidative degradation of this ferredoxin led to the formation of a stable intermediate with 1 zinc and ∼6 iron atoms. The metal centers of this intermediate were analyzed by electron paramagnetic resonance (EPR), low temperature resonance Raman, x-ray absorption, and1H NMR spectroscopies. The spectroscopic data suggest that (i) cluster II was selectively converted to a cubane [3Fe-4S]1+ cluster in the intermediate, without forming a stable radical species, and that (ii) the local metric environments of cluster I and the isolated zinc site did not change significantly in the intermediate. It is concluded that the initial step of oxidative degradation of the archaeal zinc-containing ferredoxin is selective conversion of cluster II, generating a novel intermediate containing two [3Fe-4S] clusters and an isolated zinc center. At this stage, significant structural rearrangement of the protein does not occur. We propose a new scheme for oxidative degradation of dicluster ferredoxins in which each cluster converts in a stepwise manner, prior to apoprotein formation, and discuss its structural and evolutionary implications.

X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading α-ketoglutarate dioxygenase

Abstract The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Ralstonia eutropha JMP134 is catalyzed by the α-ketoglutarate (α-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on inactive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordination with two imidazole-like ligands. Differences between the spectra for Cu TfdA and α-KG- and 2,4-D-treated samples were interpreted as a rearrangement of the g–tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal coordination environment of Cu TfdA as well as that in the active, wild-type Fe(II) enzyme. The EXAFS data are interpreted in terms of four N/O ligands (two imidazole-like) in the Cu TfdA sample and six N/O ligands (one or two imidazole-like) in the Fe TfdA sample. Addition of α-KG results in no significant structural change in coordination for Cu or Fe TfdA. However, addition of 2,4-D results in a decrease in the number of imidazole ligands in both Cu and Fe TfdA. Since this change is seen both in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenomenon. These XAS data clearly demonstrate that changes occur in the atomic environment of the metallocenter upon substrate binding.