NMR characterization of substrate binding in the phthalate dioxygenase system.

The paramagnetic enhancements in the NMR relaxation rates for the fluorine in fluorophthalates have been used to determine the position of the phthalate with respect to the mononuclear metal ion in native and metal-substituted derivatives of phthalate dioxygenase (PDO). These studies show directly that the substrate interacts with the mononuclear metal of PDO and provide the first structural characterization of this interaction. With a molecular mass of 200 kDa, PDO is one of the largest proteins studied to date by paramagnetic NMR. Two paramagnetically broadened (19)F lines were observed for monofluorophthalates bound to CoPDO. This demonstrates that fluorophthalate binds to PDO with a handedness, i.e., with the fluorine label facing to the "right" or to the "left", relative to the hyperfine tensor of the Co(II). The relative affinities of the two orientations are slightly different, with a 2-fold and 5-fold excess of the preferred orientation for 4-fluorophthalate and 3-fluorophthalate, respectively. The longitudinal relaxation rate (T(1)) and transverse relaxation rate (T(2)) data give mutually consistent fluorine to cobalt distances. These results are consistent with approximate bilateral symmetry, with the Co to 3-fluorophthalate distances ( approximately 5.5 A) approximately 25% longer than the Co to 4-fluorophthalate distances ( approximately 4. 5 A). A detailed geometric model is derived from these data. This structural characterization of the mononuclear site provides a framework to develop hypotheses for the mechanism of oxygenation by the Fe(II)-containing aromatic dioxygenases.

Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene.

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O2, CO2, and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme.(substrate or cofactor).NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O2 complexes. It is shown here that the ternary Fe(II)ACCO.ACC.NO complex is readily formed, but no Fe(II)ACCO.ascorbate.NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15N or 17O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the alpha-amino and alpha-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O2 bind to the iron as a step in O2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O2, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.

ENDOR spectroscopic evidence for the position and structure of NG-hydroxy-L-arginine bound to holo-neuronal nitric oxide synthase.

Recently, we used 35 GHz pulsed 15N ENDOR spectroscopy to determine the position of the reactive guanidino nitrogen of substrate L-arginine relative to the high-spin ferriheme iron of holo-neuronal nitric oxide synthase (nNOS) [Tierney, D. L., et al. (1998) J. Am. Chem. Soc. 120, 2983-2984]. Analogous studies of the enzyme-bound reaction intermediate, NG-hydroxy-L-arginine (NOHA), singly labeled with 15N at the hydroxylated nitrogen (denoted NR), show that NR is held 3.8 A from the Fe, closer than the corresponding guanidino N of L-Arg (4.05 A). 1,2H ENDOR of NOHA bound to holo-nNOS in H2O and D2O discloses the presence of a single resolved exchangeable proton (H1) 4.8 A from Fe and very near the heme normal. The ENDOR data indicate that NOHA does not bind as the resonance-stabilized cation in which the terminal nitrogens share a positive charge. ENDOR-determined structural constraints permit two alternate structural models for the interaction of NOHA with the high-spin heme iron. In one model, H1 is assigned to the O-H proton; in the other, it is the NR-H proton. However, the alternatives differ in the placement of the N-O bond relative to the heme iron. Thus, a combination of the ENDOR data with appropriate diffraction studies can achieve a definitive determination of the protonation state of NR and thus of the tautomeric form that is present in the enzyme-NOHA complex. The mechanistic implications of this result are further discussed.

X-ray absorption spectroscopy of the zinc site in tRNA-guanine transglycosylase from Escherichia coli.

A key step in the post-transcriptional modification of tRNA with queuine in Escherichia coli is the exchange of the queuine precursor, preQ1 into tRNA. This reaction is catalyzed by tRNA-guanine transglycosylase (TGT). We have previously shown that the E. coli TGT is a zinc metalloprotein [Chong et al. (1995) Biochemistry 34, 3694-3701]. Site-directed mutagenesis studies indicated that cysteines 302, 304, 307 and histidine 317 constitute the four ligands to the zinc. The involvement of histidine 317 is somewhat confounded by the presence of histidine 316. We have examined the zinc site in TGT (wt) and TGT (H317C) by X-ray absorption spectroscopy. The TGT (wt) data are most consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands. Interestingly, the data for TGT (H317C) are also consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands. The outer shell imidazole scattering for TGT (H317C) appears to be somewhat more ordered than that for TGT (wt), consistent with our previous suggestion that the wild-type enzyme may exist in two conformations the predominant one involving histidine 317 liganding to the zinc and the minor conformer involving histidine 316 liganding to the zinc. The minor conformer, with histidine 316 coordinating the zinc, appears to have an overall conformation that is subtly different from that of the wild-type enzyme. While TGT (H317C) has kinetic parameters very similar to the wild-type, it does not form the homotrimer quaternary structure of the wild-type. TGT (H317A) has previously [Chong et al. (1995) Biochemistry 34, 3694-3701] been found to contain a significant amount of zinc, but is essentially inactive. This suggests that careful analysis of EXAFS data can reveal subtle conformational changes in metal binding sites that are not observed in more common probes of protein conformation such as CD spectroscopy.

The zinc coordination site of the bacteriophage Mu translational activator protein, Com.

The bacteriophage Mu Com protein is a small "zinc finger-like" protein that binds a specific site in com-mom operon mRNA and activates translation of the mom open-reading-frame. Com contains six cysteine and five histidine residues that have the potential to form several alternative zinc-finger-like motifs. We have used oligonucleotide site-directed mutagenesis to individually alter each of these amino acids (Cys to Ser, and His to Asn or Gln) and tested the various forms of Com for their ability to function in vivo. We observed that mutation of any one of the four N-terminal cysteine residues (Cys-6, 9, 26 or 29) resulted in loss of Com activity. The Com protein requires zinc in order to fold into its functional tertiary structure, as demonstrated by characteristic 1H nuclear magnetic resonance (NMR) chemical shifts. 1H chemical shifts revert to random coil values in the presence of the metal chelator EDTA. The metal-binding specificity and thermal stability of Com also has been investigated using 1H NMR. We report the use of 113Cd NMR, 1H-113Cd heteronuclear spin-echo difference spectroscopy HSED and Zn extended X-ray absorption fine structure spectroscopy EXAFS to determine the zinc/protein stoichiometry as 1:1 and the ligand environment as tetrathiolate. Comparative NMR spectra of Com mutants C6S and C39S suggest position 6 is involved in zinc coordination, while position 39 is not metal-liganded. These studies indicate that the metal coordination, site of Com is a four-cysteine complex, involving residues 6, 9, 26 and 29.

X-ray absorption spectroscopy of the iron site in Escherichia coli Fe(III) superoxide dismutase.

The local structure of the iron site in ferric superoxide dismutase from Escherichia coli has been characterized by X-ray absorption spectroscopy. In the resting state of the enzyme at pH 7.0, the iron is five-coordinate with an average metal-ligand bond length of 1.98 A. Binding of azide causes a reduction in the intensity of the bound state 1s-->3d transition and an increase of 0.08 A in average bond length. Both are indicative of an increase in the iron coordination number. Raising the pH from 7.0 to 10.5 causes a similar 0.08 A increase in the average bond length, again suggesting an increase in the iron coordination number. At intermediate pH (9.4), the average bond length is 2.03 A, consistent with an approximately 50:50 mixture of the limiting high and low pH forms. Similarly, the absorption edge structure varies continuously from pH 7 to 10.5. These spectra can be fit to a titration curve with a pKa of approximately 9.8. These data suggest that the pH-dependent transition, previously identified by UV-vis, EPR, and activity measurements, may be the conversion of the iron from five- to six-coordinate, presumably through coordination by hydroxide. The 1s-->3d transition for ferric superoxide dismutase at high pH is broader but not significantly less intense than that at pH 7. This suggests that the high pH form may be significantly distorted from octahedral symmetry. At pH 7, the ferric and ferric + azide samples undergo slow X-ray induced photoreduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiol ligation of two zinc atoms to a class I tRNA synthetase: evidence for unshared thiols and role in amino acid binding and utilization.

Class I tRNA synthetases generally contain a characteristic N-terminal catalytic core joined to a C-terminal domain that is idiosyncratic to the enzyme. The closely related class I Escherichia coli methionyl- and isoleucyl-tRNA synthetases each have a single zinc atom coordinated to ligands contained in the catalytic domain. Isoleucyl-tRNA synthetase has a second, functionally essential, zinc bound to ligands at the C-terminal end of the 939 amino acid polypeptide. Recent evidence suggested that this structure curls back and interacts directly or indirectly with the active site. We show here by X-ray absorption spectroscopy that the average Zn environment contains predominantly sulfur ligands with a Zn-S distance of 2.33 A. A model with eight coordinated thiolates divided between two Zn(Cys)4 structures best fit the data which are not consistent with a thiolate-bridged Zn2(Cys)6 structure joining the C-terminal end with the N-terminal active site domain. We also show that zinc bound to the N-terminal catalytic core is important specifically for amino acid binding and utilization, although a direct interaction with zinc is unlikely. We suggest that, in addition to idiosyncratic sequences for tRNA acceptor helix interactions incorporated into the class-defining catalytic domain common to class I enzymes, the architecture of at least some parts of the amino acid binding sites may differ from enzyme to enzyme and include motifs that bind zinc.