Metalloprotein Structure Research Articles

Global mapping of structural solutions provided by the extended X-ray absorption fine structure ab initio code FEFF 6.01: structure of the cryogenic photoproduct of the myoglobin-carbon monoxide complex.

X-ray methods based on synchrotron technology have the promise of providing time-resolved structural data based on the high flux and brightness of the X-ray beams. One of the most closely examined problems in this area of time-resolved structure determination has been the examination of intermediates in ligand binding to myoglobin. Recent crystallographic experiments using synchrotron radiation have identified the protein tertiary and heme structural changes that occur upon photolysis of the myoglobin--carbon monoxide complex at cryogenic temperatures [Schlichting, I., Berendzen, J., Phillips, G., & Sweet, R. (1994) Nature 371, 808--812]. However, the precision of protein crystallographic data (approximately 0.2 A) is insufficient to provide precise metrical details of the iron--ligand bond lengths. Since bond length changes on this scale can trigger reactivity changes of several orders of magnitude, such detail is critical to a full understanding of metalloprotein structure--function relationships. Extended X-ray absorption fine structure (EXAFS) spectroscopy has the potential for analyzing bond distances to a precision of 0.02 A but is hampered by its relative insensitivity to the geometry of the backscattering atoms. Thus, it is often unable to provide a unique solution to the structure without ancillary structural information. We have developed a suite of computer programs that incorporate this ancillary structural information and compute the expected experimental spectra for a wide ranging series of Cartesian coordinate sets (global mapping). The programs systematically increment the distance of the metal to various coordinating ligands (along with their associated higher shells). Then, utilizing the ab initio EXAFS code FEFF 6.01, simulated spectra are generated and compared to the actual experimental spectra, and the differences are computed. Finally, the results for hundreds of simulations can be displayed (and compared) in a single plot. The power of this approach is demonstrated in the examination of high signal to noise EXAFS data from a photolyzed solution sample of the myoglobin--carbon monoxide complex at 10 K. Evaluation of these data using our global mapping procedures placed the iron to pyrrole nitrogen average distances close to the value for deoxymyoglobin (2.05 +/- 0.01 A), while the distance from iron to the proximal histidine nitrogen is seen to be 2.20 +/- 0.04 A. It is also shown that one cannot uniquely position the CO ligand on the basis of the EXAFS data alone, as a number of reasonable minima (from the perspective of the EXAFS) are observed. This provides a reasonable explanation for the multiplicity of solutions that have been previously reported. The results presented here are seen to be in complete agreement with the crystallographic results of Schlichting et al. (1994) within the respective errors of the two techniques; however, the extended X-ray absorption fine structure data allow the iron--ligand bond lengths to be precisely defined. An examination of the available spectroscopic data, including EXAFS, shows that the crystallographic results of Schlichting et al. (1994) are highly relevant to the physiological solution state and must be taken into account in any attempt to understand the incomplete relaxation process of the heme iron for the Mb*CO photoproduct at low temperature.

Analysis of Local Structure of Metalloproteins by Fluorescent XAFS Method

Principle of XAFS (X-ray Absorption Fine Structure) was briefly reviewed in relation to its application to metalloproteins in biological systems. Our experiences in PF of KEK for these ten years were also summarized toward the 3rd generation photon ring.

Assignment of Pseudo‐contact‐shifted 1H NMR resonances in the EF site of Yb3 +‐substituted rabbit parvalbumin through a combination of 2D techniques and magnetic susceptibility tensor determination

AbstractPseudo‐contact‐shifted 1H NMR resonances of rabbit parvalbumin substituted with Yb3+ at the Ca2+ EF site have been sequence‐specific assigned by a combination of 2D techniques and magnetic susceptibility tensor determination. By taking the X‐ray structure of the homologous carp protein as a starting point, the sequence‐specific assignment proceeded in a stepwise fashion. The first resonances assigned on the basis of their unique connectivity pattern were used to make a first coarse determination of the X‐tensor parameters; the latter were used to guess the identity of further hyperfine‐shifted resonances, which were then checked through 2D connectivities and in turn allowed us to improve the reliability of the X‐tensor parameters. This cyclic procedure was repeated until virtually all pseudo‐contact‐shifted resonances were assigned. In the last steps the X‐tensor parameters did not change appreciably. The orientation of the tensor was found to be very similar to that previously proposed for the homologous Yb3+−substituted protein from carp. Modest discrepancies between some calculated and experimental shift values in the present system were found to be due mainly to structural differences between the present protein and the homologous protein whose x‐ray structure was used as model and, to a lesser extent, to indetermination in the diamagnetic shift values. The success of the analysis and the univocal solution for the X‐tensor parameters under these conditions show that the above procedure is robust enough to permit both signal assignment and X‐tensor determination even when the structural data are only a rough approximation of the actual structure. These findings may open the way to the development of a systematic use of pseudo‐contact shifts as constraints for MD refinement of solution structures of paramagnetic metalloproteins.

26] Detecting metal-metal interactions and measuring distances between metal centers in metalloproteins

Publisher Summary Even if a high-resolution structure of a metalloprotein is not available, it is possible to obtain important insights into molecular structure and function through the application of appropriate biophysical techniques. This chapter addresses one particular aspect of studying the structures of metalloproteins in solution—namely, methods and techniques to characterize the organization of metal centers in multimetal proteins. Before proceeding to the discussion of methods, a short account of the structural and functional variety of closely spaced metal atoms in multimetal proteins is given. Metals in these proteins either form well separated mononuclear complexes or are closely spaced or clustered. Metal-metal interactions occur within one protein subunit, between subunits, or between a metal cofactor or a label and an intrinsic metal atom. In this chapter, attention is focused on methods that can detect directly (physically) interacting metal atoms and that can measure distances between them. Accordingly, interaction becomes largely synonymous with proximity throughout this chapter, except that the few existing methods that can measure longer distances are also presented. Binuclear arrangements of metal ions are most frequently recognized and higher nuclearity is also observed.

11] Infrared spectroscopy

Publisher Summary This chapter discusses the way metal ion environments in metalloproteins and metalloenzymes can be studied by using infrared (IR) spectroscopy. It explains experimental techniques and methods of analysis and interpretation by using selected examples. Fourier transform (FT) IR spectroscopy is mentioned only briefly. Although both IR and Raman spectroscopies provide information about transitions between two vibrational states, there are several advantages and disadvantages unique to each. IR and Raman spectroscopy are complementary, and both spectra should be obtained whenever possible. In fact, vibrational spectra of most of the biological compounds discussed in this chapter have been studied by IR as well as Raman spectroscopy. This chapter discusses IR spectra of axial ligands (CO, NO, O 2 , CN - , and N 3 - ), which are bound to metalloproteins and metalloenzymes. IR spectra of metalloproteins and metalloenzymes themselves constitute another area that can be studied with FTIR spectroscopy, but their full potential as a probe of the structure of metal-containing biological systems has yet to be established. In this respect, there is no doubt that Raman spectroscopic techniques have proved to be much more informative. Nevertheless, several promising FTIR studies, particularly on the secondary structures of metalloproteins, have been carried out.

2] Cadmium-113 nuclear magnetic resonance applied to metalloproteins

Publisher Summary Among the naturally occurring isotopes of cadmium there are two, 113 Cd and 111 Cd, that have a nuclear spin of ½ and are useful as nuclear magnetic resonance (NMR) probes for cadmium-containing molecules. Both isotopes are present at a significant percent natural abundance, namely, 12.75 and 12.26% for 111 Cd and 113 Cd, respectively. They are both obtainable in 96% purity. Although there is only one naturally occurring well-characterized protein, metallothionein, that accumulates cadmium, most metalloproteins containing the Zn 2+ ion—5 d 10 electron configuration—can be substituted with the heavier Cd 2+ ion (6 d 10 ). In the case of metalloenzymes, the Cd substitution has often led to a protein that maintains measurable catalytic activity. For a variety of Zn-containing DNA-binding proteins that require Zn to maintain the fold required for DNA recognition and binding, the Cd-substituted proteins have retained DNA binding. Thus, from both structural and functional standpoints 113 Cd ( 111 Cd) NMR can be used as a valuable probe of zinc metalloprotein structure and function. The cadmium(II) ion has an ionic radius of 0.97 A nearly the same as that of Ca 2+ , 0.99 A (Pauling radii). Thus, Cd 2+ can be substituted at most Ca 2+ binding sites found in proteins. The Cd 2+ ion can also fit the sites occupied by the Cu + ion (5 d 10 ) and thus can be used to probe the metal sites in copper electron transfer proteins in which copper alternates between the Cu + and Cu 2+ states. Metal ion replacement employs anaerobic techniques under which the Cu + ion is removed and replaced by 113 Cd 2+ . This chapter describes the application of 113 Cd NMR methods to the study of metalloproteins.

The prediction and characterization of metal binding sites in proteins.

The rational engineering of novel functions into proteins can only be attempted when the underlying structural scaffold on which the new function is displayed and the structure of the target protein are both well understood. To introduce functions mediated by metals it is therefore necessary to identify the principal liganding residues for the chosen metal, the required architecture of the metal-ligand complex and sites within the target protein that could accommodate such sites. Here we present a method that applies structural information from the protein data bank to the ab initio design and characterization of novel metal binding sites. The prediction method has been tested on 28 metalloprotein structures from the Brookhaven Protein Data Bank. It successfully identified > 90% of the metal binding sites. In addition, we have used the method to design and characterize zinc binding sites in two antibody structures. Metal binding studies on one of these putative metalloantibodies showed metal binding, confirming the predictive power of the method.

Metal-binding sites in proteins.

A dramatic increase in the number of solved metalloprotein structures and recent breakthroughs in structural analysis have provided a sufficiently detailed understanding of the structural chemistry of some metal-binding sites to allow successful design. As a result, metal-binding site design is now one of the most powerful and promising approaches for influencing protein folding, assembly, stability and catalysis.

Chirality and Isomerism in Binuclear Iron Complexes with Sulfur Ligands: [Fe(CO)(μ‐“S4”)]2, a Model Complex for Oxidoreductases

Metal atoms at the active centers of oxidoreductases must be chirotopic. In the course of a search for model complexes for nitrogenases and hydrogenases, a new type of chiral complex was discovered, represented by the complex 1. It was obtained by coupling two homochiral [Fe(CO)(“S4”)] fragments, and it contains Fe centers that are capable of binding σ-π ligands. This complex may thus be regarded as one segment of a metalloprotein structure. (“S4”)2⊖ = 2,2′-(ethylenedithio)dibenzene thiolate.

Transcription factor IIIA induced bending of the Xenopus somatic 5S gene promoter.

Transcription factor IIIA (TFIIIA), the canonical zinc-finger protein, is a protein of relative molecular mass 39,000 (39K) that is required for transcription of 5S-ribosomal subunit genes in Xenopus. It binds in a sequence-specific manner to the internal control region of the 5S gene (see Fig. 1) and facilitates transcription of the gene by RNA polymerase III. It also binds to the 5S gene product to form a 7S ribonucleoprotein particle. In oocytes the 7S particle acts as a storage form of the RNA to be utilized later in development. TFIIIA binds to DNA through its 30 K N-terminal domain, which contains nine zinc-fingers. TFIIIA was the first protein described to have this type of DNA binding motif, but numerous other proteins have now been shown to have zinc-finger domains. A structure for a single zinc-finger from the yeast protein ADR1, was recently proposed based on two-dimensional NMR data (ref. 8), and a similar structure was proposed based on comparison with crystal structures of other metalloproteins. Although models for the interaction of TFIIIA with the 5S-ribosomal gene DNA have been proposed, based on nuclease digestion and methylation interference data, little precise structural information is available for TFIIIA and the physical basis for the interaction of zinc-fingers with DNA is not understood. Using both circular permutation and circularization assays we provide convincing biochemical evidence that TFIIIA bends the DNA at the internal promoter of the 5S gene.

Zinc fingers: Gilt by association

A major focus of molecular biology is the identification of mechanisms by which eukaryotic gene transcription is regulated in a precise temporal and spatial fashion. The identification of transcriptional factors with the ability to recognize and bind to specific DNA sequences has provided important clues to elucidating these mechanisms. X-ray crystallographic analysis of prokaryotic DNA binding proteins such as lambda repressor and cro revealed a common helix-turn-helix motif responsible for their interaction with DNA(Pabo and Sauer, Ann. Rev. Biothem. 53, 293-321, 1984). The notion that this structure is universally used for specific DNA interaction was challenged by Klug’s recognition of a new DNA binding motif in Xenopus laevis transcription factor TFIIIA: the zinc finger (Miller et al., EMBO J. 4, 1609-1614, 1985). Although originally recognized in an RNA polymerase Ill transcription factor, recent analysis has revealed potential metal binding fingers as a surprisingly common structure in a variety of nucleic acid binding proteins (for review see Berg, Science 232,485-487, 1986), including factors likely to influence polymerase II transcription. Finger Structure and Metal Binding Isolation and comparison of cDNAs encoding potential finger proteins indicate the existence of at least two classes of proteins with specific nucleic acid recognition capabilities. These classes are characterized according to the number and position of the cysteine and histidine residues available for zinc coordination (see table). The &HZ class is typified by TFIIIA and is comprised of proteins containing, as the basic structural unit, pairs of cysteines and histidines separated by a loop of twelve amino acids. Although most easily visualized as a finger of amino acids anchored by zinc and projecting from the surface of the protein (see figure, part A), an alternate view, inferred from structures of known metalloproteins, suggests the more complex model depicted in part B of the figure (Berg, PNAS 85, 99-102, 1988). In the C2H2 proteins identified, the finger motif is tandemly repeated a minimum of two times with a 7 to 8 amino acid linker separating the units (the H/C link). In contrast to the C2H2 group, the C, proteins have a variable number of conserved cysteines available for metal chelation. For the yeast transcriptional factors, such as GAL4, the putative finger region exists as a cluster of six invariant cysteines (C, family), whereas the steroid receptors contain two apparently unrelated fingers encoded by separate exons (Huckaby et al., PNAS 84, 8380-8384, 1987) with four (C,) and five (C,) conserved Minireview

Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain

Transcription factor Sp1 is a protein present in mammalian cells that binds to GC box promoter elements and selectively activates mRNA synthesis from genes that contain functional recognition sites. We have isolated a cDNA that encodes the 696 C-terminal amino acid residues of human Sp1. By expression of truncated fragments of Sp1 in E. coli, we have localized the DNA binding activity to the C-terminal 168 amino acid residues. In this region, Sp1 has three contiguous Zn(II) finger motifs, which are believed to be metalloprotein structures that interact with DNA. We have found that purified Sp1 requires Zn(II) for sequence-specific binding to DNA. Thus, it is likely that Sp1 interacts with DNA by binding of the Zn(II) fingers. To facilitate the identification of mutant variants of Sp1 that are defective in DNA binding, we have also devised a bacterial colony assay for detection of Sp1 binding to DNA.

The tertiary structure of azurin from Pseudomonas denitrificans as determined by Cu resonant diffraction using synchrotron radiation

The structure of Pseudomonas denitrificans azurin has been solved at 3.0 Å resolution by film data collection methods using synchrotron radiation. Bijvoet pairs of reflections were collected using 8.98 keV radiation where both Δ f′ and Δ f″, the real and imaginary corrections to azurin's Cu prosthetic group scattering, respectively, are large, and using 8.00 keV radiation where these corrections are smaller. The Cu atom was located by difference Patterson syntheses and used to phase the observed protein structure-factor moduli of selected reflections. An atomic model of the protein was built using a restricted data set and phases for all observed reflections to 3.0 Å resolution were subsequently calculated from the initial model. The atomic model was subsequently rebuilt using all observed data. The results of this work are presented here and illustrate the utility of synchrotron radiation phasing techniques in solving the structures of metalloproteins without recourse to multiple isomorphous replacement.

Evidence for a metalloprotein stucture of plasma membrane 5' -nucleotidase

To point out the metalloprotein structure of bovine liver plasma membrane 5'-nucleotidase we studied the inhibition mechanism of the purified enzyme by EDTA: this apparently non-competitive inhibition seems to be dependent on EDTA concentration, pH, temperature and incubation time. When the restoration of activity was assayed by addition of divalent cations or by gel filtration, the inhibition became progressively irreversible with time. Incubation of the enzyme with [ 14C]EDTA allowed us to observe, after gel filtration as well as after sucrose gradient ultracentrifugation, that the chelating agent is bound to 5'-nucleotidase.

A program for the calculation of X-ray reflection intensities

The structure of Pseudomonas denitrificans azurin has been solved at 3.0 Å resolution by film data collection methods using synchrotron radiation. Bijvoet pairs of reflections were collected using 8.98 keV radiation where both Δf′ and Δf″, the real and imaginary corrections to azurin's Cu prosthetic group scattering, respectively, are large, and using 8.00 keV radiation where these corrections are smaller. The Cu atom was located by difference Patterson syntheses and used to phase the observed protein structure-factor moduli of selected reflections. An atomic model of the protein was built using a restricted data set and phases for all observed reflections to 3.0 Å resolution were subsequently calculated from the initial model. The atomic model was subsequently rebuilt using all observed data. The results of this work are presented here and illustrate the utility of synchrotron radiation phasing techniques in solving the structures of metalloproteins without recourse to multiple isomorphous replacement.

Local coordination geometry in Fe and Ca metalloproteins by XANES using synchrotron radiation

X-ray absorption spectroscopy using synchrotron radiation is opening new perspectives in the study of fundamental biological problems of structure and function of metalloproteins because of its unique capability to give direct structural information on the metal active sites of proteins in solution. The direct structural information in X-ray absorption spectra arise because of their interpretation in terms of electron scattering by neighbour atoms of the electrons photoemitted at the metal site. EXAFS oscillations arising from single scattering processes can give distances, coordination numbers and the type of neighbour atoms! A recent development on the X-ray absorption near edge structure: XANES [1] extending over about 50 eV energy range demonstrates that this part of the X-ray absorption spectrum also can be interpreted in terms of electron scattering by neighbour atoms. The spectra show strong multiple scattering resonances, like low energy electron spectroscopy, determined by atomic geometrical distribution of the neighbour atoms. The actual ‘state of art’ is such that after distance determination by EXAFS, XANES is able to distinguish between different possible geometrical structures [2]. We report XANES spectra of hemoglobin and related heme-proteins where we have found evidence of: 1. Fe displacement relative to the porphyrin plane which cannot be determined by other methods in solution. 2. Variation of the ligand bonding angle FeCO and FeCN in hemoglobin in solution where CO is titled and CN is vertical relative to the porphyrin plane. 3. Time resolved XANES can provide information on the dynamics of atoms in the active sites during protein function. In calcium proteins [4, 5] troponin-C and calmodulin [6] different orientations of COO − groups of aspartic and glutamic residues bound to Ca 2+ in the EF-hand loop can be distinguished and different sites are identified. The presence of the allosteric role of Mg 2+, and the effects of drugs on calmodulin have been found.

Susceptibility and structure of metallo-proteins and model systems

The interest in the study of the diamagnetic properties of the whole molecule and/or the magnetic moment of metal ions bound in relation to the structure and function of proteins is briefly outlined. The advantages in using high-resolution superconducting magnetometers are described together with the main features of the experimental technique and its foreseen advancement. A few examples, as representative of classes of problems, are discussed in more detail with reference to recent experimental studies.

Synchrotron radiation: new window on metalloprotein structure.

Synchrotron radiation: new window on metalloprotein structure.

A short range probe for investigating metalloprotein structures: Fourier analysis of the extended x-ray absorption fine structure

A short range probe for investigating metalloprotein structures: Fourier analysis of the extended x-ray absorption fine structure

Iron and Free Radical in Ribonucleotide Reductase: EXCHANGE OF IRON AND MÖSSBAUER SPECTROSCOPY OF THE PROTEIN B2 SUBUNIT OF THE ESCHERICHIA COLI ENZYME

Abstract Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits (Protein B1 and Protein B2), both required for activity. Protein B2 contains 2 iron atoms and a lesser, variable amount of a paramagnetic species, characterized by g = 2.0047 EPR and 410 nm electronic absorptions. Except for the 410 nm band, Protein B2 much resembles oxy- and methemerythrins in electronic spectra. The paramagnetic species is probably organic free radical rather than metal, is dependent upon the presence of iron in Protein B2, is directly correlated with enzymatic activity, and is destroyed by NH2OH and hydroxyurea (Ehrenberg, A., and Reichard, P. (1972) J. Biol. Chem. 247, 3485–3488). We here describe complete removal of iron from Protein B2, reconstitution to active metalloenzyme, and Mossbauer spectroscopy of [57Fe]Protein B2. Addition of excess Fe2+ to metal-free protein yielded reconstituted/reactivated protein with 2:1 iron:subunit stoichiometry and a hemerythrinlike electronic spectrum, but with 120 to 390% of the original specific enzymatic activity and correspondingly increased 410 nm and EPR absorptions. NH2OH- or hydroxyurea-inactivated protein was also converted to highly active enzyme subunit by the same iron substitution procedures. Mossbauer and electronic spectra suggested that Protein B2 contains 2 nonidentical high spin Fe(III) ions in an antiferromagnetically coupled binuclear complex that resembles both methydroxohemerythrin and oxyhemerythrin. Below 195°K the iron in Protein B2 was completely diamagnetic, in confirmation of the previous assignment of the 410 nm- and EPR-absorbing species as stable organic free radical of unknown structure. We propose that the function of iron in Protein B2 is the initial generation of radical from a protein-bound group, and that stability of the radical depends upon some continuing interaction with the iron center. The radical is apparently less stable than is metalloprotein structure: this would account for the variability of radical content in native protein and for the g 100% yield of radical obtainable by substitution of the iron. We further propose that the free radical participates in the reduction of ribonucleotides by the enzymatically active Protein B1-Protein B2 complex.