Thiolate ligation of the active site Fe2+ of isopenicillin N synthase derives from substrate rather than endogenous cysteine: spectroscopic studies of site-specific Cys----Ser mutated enzymes.

Isopenicillin N synthase (IPNS) catalyzes double ring closure of the tripeptide (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of penicillin-type antibiotics. Our previous spectroscopic study using IPNS from Cephalosporium acremonium expressed in Escherichia coli [Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681] indicated that a thiolate enters the coordination of the essential active site Fe2+ when ACV binds to IPNS. The presence of an Fe-S bond in the IPNS.ACV complex is confirmed by EXAFS data presented in the preceding paper [Scott, R. A., Wang, S., Eidsness, M. K., Kriauciunas, A., Frolik, C. A. & Chen, V. J. (1992) Biochemistry (preceding paper in this issue)]. However, these studies leave unclear whether the coordinating thiolate derives from ACV or an endogenous cysteine. Here, we examine the spectroscopic properties of three genetically engineered variants of IPNS in which the only two endogenous cysteines are individually and collectively replaced by serine. The EPR, M?ssbauer, and optical spectra of the mutant enzymes and their complexes with ACV, NO, or both ACV and NO are found to be essentially the same as those of wild-type IPNS, showing that the endogenous cysteines are not Fe2+ ligands in any of these complexes. Spectral quantitations show that the double Cys----Ser mutation decreases the affinity of the enzyme for ACV by about 6-fold, suggesting that the endogenous cysteines influence the structure of the substrate binding pocket remote from the iron. Thiolate complexation of the Fe2+ is also examined using ACV analogues. All ACV analogues examined in which the cysteinyl thiol moiety is unaltered are found to bind to the IPNS.NO complex to give optical and EPR spectra very similar to those of the ACV complex. In contrast, analogues in which the cysteinyl moiety of ACV is replaced with serine or cysteic acid fail to elicit the characteristic EPR and optical features despite the fact that they are bound with reasonable affinity to the enzyme. These results demonstrate that the thiolate of ACV coordinates the Fe2+. The EPR spectra of both the IPNS.NO and IPNS.ACV.NO complexes are broadened for samples prepared in 17O-enriched water, showing that water (or hydroxide) is also an iron ligand in each case. Thus, the Fe2+ coordination of the IPNS.ACV.NO complex accommodates at least three exogenous ligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for Fe-S interaction in the enzyme-substrate complex.

Isopenicillin N synthase from Cephalosporium acremonium (IPNS; M(r) 38.4K) is an Fe(2+)-requiring enzyme which catalyzes the oxidative conversion of (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, with concomitant reduction of O2 to 2H2O. Chemical and spectroscopic data have suggested that catalysis proceeds via an enzyme complex of ACV bound to the iron through its cysteinyl thiolate [Baldwin, J. E., & Abraham, E. P. (1988) Nat. Prod. Rep. 5, 129-145; Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. Here we have employed the technique of Fe K-edge extended X-ray absorption fine structure (EXAFS) to characterize the iron site and to seek direct evidence for or against the formation of an Fe-S interaction upon ACV binding. Our data collected in the absence of substrate and O2 are consistent with the iron center of IPNS being coordinated by only (N,O)-containing ligands in an approximately octahedral arrangement and with an average Fe-(N,O) distance of 2.15 +/- 0.02 A. Upon anaerobic binding of ACV, the iron coordination environment changes considerably, and the associated Fe EXAFS cannot be adequately simulated without incorporating an Fe-S interaction at 2.34 +/- 0.02 A along with four or five Fe-(N,O) interactions at 2.15 +/- 0.02 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear Overhauser effect study of the heme active site structure of chloroperoxidase.

Chloroperoxidase, a glycoprotein from the mold Caldariomyces fumago, has been investigated in its ferric low-spin cyanide-ligated form through use of nuclear Overhauser effect (NOE) spectroscopy to provide information on the heme pocket electronic/molecular structure. Spin-lattice relaxation times for the hyperfine-shifted heme resonances were found to be three times less than those in horseradish peroxidase. This must reflect a slower electronic relaxation rate for chloroperoxidase than for horseradish peroxidase as a consequence of axial ligation of cysteine in the former versus histidine in the latter enzyme. Isoenzymes A1 and A2 of chloroperoxidase show the largest chemical shift differences near the heme propionate on the basis of NOE measurements. This suggests that the primary structure differences for the two isoenzymes are communicated to the heme group through the ring propionate substituents. A downfield peak has been detected in chloroperoxidase with chemical shift, T1, and line width characteristics similar to those of the C epsilon-H proton of the distal histidine residue. The NOE pattern and T1's of the peaks in the 0.0 to -5.0 ppm upfield region are consistent with the presence of an arginine amino acid residue in the heme pocket near either the 1-CH3 or 3-CH3 group. Existence of catalytically important distal histidine and arginine amino acid residues in chloroperoxidase shows it to be structurally similar to peroxidases rather than to the often compared monooxygenase, cytochrome P-450. This result supports the earlier conclusions of Sono et al. [Sono, M., Dawson, J.H., Hall, K., & Hager, L.P. (1986) Biochemistry 25, 347-356].     

Structural studies on the reaction of isopenicillin N synthase with the truncated substrate analogues delta-(L-alpha-aminoadipoyl)-L-cysteinyl-glycine and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alanine

Isopenicillin N synthase (IPNS), a non-heme iron(II)-dependent oxidase, catalyzes conversion of the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN), concomitant with the reduction of dioxygen to two molecules of water. Incubation of the "truncated"substrate analogues delta-(l-alpha-aminoadipoyl)-l-cysteinyl-glycine (ACG) and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-alanine (ACA) with IPNS has previously been shown to afford acyclic products, in which the substrate cysteinyl residue has undergone a two-electron oxidation. We report X-ray crystal structures for the anaerobic IPNS/Fe(II)/ACG and IPNS/Fe(II)/ACA complexes, both in the absence and presence of the dioxygen analogue nitric oxide. The overall protein structures are very similar to those of the corresponding IPNS/Fe(II)/ACV complexes; however, significant differences are apparent in the vicinity of the active site iron. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-terminal carboxylate of this substrate is oriented toward the active site iron atom, apparently hydrogen-bonded to an additional water ligand at the metal; this is a different binding mode to that observed in the IPNS/Fe(II)/ACV complex. ACA binds to the metal in a manner that is intermediate between those observed for ACV and ACG. The addition of NO to these complexes initiates conformational changes such that both the IPNS/Fe(II)/ACG/NO and IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These results further demonstrate the feasibility of metal-centered rearrangements in catalysis by non-heme iron enzymes and provide insight into the delicate balance between hydrophilic-hydrophobic interactions and steric effects in the IPNS active site.     

Point mutations at the type I Cu ligands, Cys457 and Met467, and at the putative proton donor, Asp105, in Myrothecium verrucaria bilirubin oxidase and reactions with dioxygen

The type I Cu site in the Cys457Ser mutant of Myrothecium verrucaria bilirubin oxidase was vacant, but the trinuclear center composed of a type II Cu and a pair of type III Cu's was fully occupied by three Cu ions. Cys457Ser could react with dioxygen, affording reaction intermediate I with absorption maxima at 340, 470, and 675 nm. This intermediate corresponds to that obtained from laccase, whose type I Cu is cupric and type II and III Cu's are cuprous [Zoppellaro, G., Sakurai, T., and Huang, H. (2001) J. Biochem. 129, 949-953] or whose type I Cu is substituted with Hg [Palmer, A. E., Lee, S. K., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 6591-6599]. Another type I Cu mutant, Met467Gln, with modified spectroscopic properties and redox potential, afforded reaction intermediate II with absorption maxima at 355 and 450 nm. This intermediate corresponds to that obtained during the reaction of laccase [Sundaram, U. M., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 12525-12540; Huang, H., Zoppellaro, G., and Sakurai, T. (1999) J. Biol. Chem. 274, 32718-32724]. According to a three-dimensional model of bilirubin oxidase, Asp105 is positioned near the trinuclear center. Asp105Glu and Asp105Ala exhibited 46 and 7.5% bilirubin oxidase activity compared to the wild-type enzyme, respectively, indicating that Asp105 conserved in all multi-copper oxidases donates a proton to reaction intermediates I and II. In addition, this amino acid might be involved in the formation of the trinuclear center and in the binding of dioxygen based on the difficulties in incorporating four Cu ions in Asp105Ala and Asp105Asn and their reactions with dioxygen.     

1H NMR and NOE studies of the purple acid phosphatases from porcine uterus and bovine spleen.

The diiron active sites of the purple acid phosphatases from porcine uterus (also called uteroferrin, Uf) and bovine spleen (BSPAP) and their complexes with tungstate are compared by 1H NMR and NOE techniques. The paramagnetically shifted features of the 1H NMR spectrum of reduced BSPAP are similar to those of reduced Uf, while the spectra of the tungstate complexes are almost identical. These observations suggest that the two active sites are quite similar, in agreement with the greater than 90% sequence homology found in the two enzymes. Nuclear Overhauser effect (NOE) experiments on the His N-H resonances show that the Fe(III)-His residue is N epsilon-coordinated, while the Fe(II)-His is H delta-coordinated in both enzymes. On the basis of the above NMR and NOE results, our previously proposed model for the dinuclear iron active site of Uf [Scarrow, R. C., Pyrz, J. W., & Que, L., Jr. (1990) J. Am. Chem. Soc. 112, 657-665] is corroborated, refined, and found to represent the diiron center of BSPAP as well.     

Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c.

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3-CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso-1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the beta-CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3-CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870-7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3-CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 beta-CH2 group and the heme 3-CH3 substituent. In addition, studies of the effect of pH on the 1H-NMR spectrum of yeast iso-1 ferricytochrome c show that the heme 3-CH3 proton resonance exhibits a pH-dependent shift with an apparent pK in the range of 6.0-7.0. The chemical shift change of the yeast iso-1 ferricytochrome c heme 3-CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934-4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C-terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3-CH3 group in yeast iso-1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8-CH3 and the 7 alpha-CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of approximately 7.0. The titrating group responsible for these spectral changes is proposed to be His39.     

Spectroscopic evidence for an intermediate in the T6 to R6 allosteric transition of the Co(II)-substituted insulin hexamer.

The phenol-induced conformational transition in the insulin hexamer is known to involve a large change in structure wherein residues 1-8 of the insulin B-chain are transformed from an extended coil (T-state) to a helix (R-state). This change in protein conformation both exposes a cryptic protein pocket on each subunit to which phenol binds and forces the HisB10 zinc sites to undergo a change in coordination geometry from octahedral to tetrahedral [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 593-596]. Substitution of Co(II) for Zn(II) at the HisB10 sites introduces a sensitive chromophoric probe of the structural and chemical events that occur during this allosteric transition [Roy, M., Brader, M. L., Lee, R. W.-K., Kaarsholm, N. C., Hansen, J. F., & Dunn, M. F. (1989) J. Biol. Chem. 264, 19081-19085]. In this study, using rapid-scannig stopped-flow (RSSF) UV-visible spectroscopic studies, we demonstrate that a transient chemical intermediate is formed during the phenol-induced conversion of Co(II)-substituted hexamer from the T-state to the R-state. Decomposition of the RSSF spectra gave a spectrum for the intermediate with d-d transitions consistent with the assignment of the intermediate as either a distorted tetrahedral or a 5-coordinate Co(II) species. Possible structures for the intermediate and the implications of these findings to the allosteric mechanism are considered.     

ADP-glucose pyrophosphorylase from potato tuber: site-directed mutagenesis of homologous aspartic acid residues in the small and large subunits

Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.A. and Preiss J. (2001) J. Biol. Chem., 276, 46319-46325]. The residue is highly conserved throughout the family of ADP-Glc PPases, as well as throughout the super-family of sugar-nucleotide pyrophosphorylases. In the heterotetrameric ADP-Glc PPase from potato (Solanum tuberosum L.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP-Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP-Glc PPase from potato tuber, we used site-directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP-Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3-phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.     

Prediction of q-values and conformations of gadolinium chelates for magnetic resonance imaging

For gadolinium chelates, we determined that there is a linear correlation between calculated solvent-accessible surface area and q-value, the number of rapidly exchanging water molecules directly bound to the gadolinium ion. A calibration curve was developed to predict q-value based on the solvent-accessible surface area of gadolinium. This predictive method was validated with the following gadolinium crystal structures: (ethylenediaminetetraacetic acid)-gadolinium(III) [Gd(EDTA)] [Templeton, L. K., Templeton, D. H., Zalkin, A., and Ruben, H. W. (1982) Anomalous Scattering by Praseodymium, Samarium, and Gadolinium and Structures of their Thylenediaminetetraacetate (EDTA) Salts. Acta Crystallogr., Sect. B 38, 2155], (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid)-gadolinium(III) [Gd(DOTA)] [Dubost, J.-P., Leger, J.-M., Langlois, M.-H., Meyer, D., and Schaefer, M. (1991) Structure of a Magnetic Resonance Imaging Agent - The Gadolinium-DOTA Complex C(16)H(24)N(4)O(8)NaGd, 5H(2)O. C. R. Acad. Sci., Ser. 2 312, 349], (diethylenetriaminepentaacetic acid)-gadolinium(III) [Gd(DTPA)] [Stezowski, J. J., and Hoard, J. L. (1984) Heavy Metal Ionophores - Correlations Among Structural Parameters of Complexed Nonpeptide Polyamino Acids. Isr. J. Chem. 24, 323], (diethylenepenta-acetato)-gadolinium(III) [Gd(DTPA-BEA)] [Smith, P. H., Brainard, J. R., Morris, D. E., Jarvinen, G. D., and Ryan, R. R. (1989) Solution and Solid-State Characterization of Europium and Gadolinium Schiff-Base Complexes and Assessment of their Potential as Contrast Agents in Magnetic Resonance Imaging. J. Am. Chem. Soc. 111, 7437], and (1,7,13-triaza-4,10, 16-trioxacyclo-octadecane-N,N',N' '-triacetato)-gadolinium(III) [Gd(TTTA)] [Chen, D., Squattrito, P. J., Martell, A. E., and Clearfield, A. (1990) Synthesis and Crystal Structure of a 9-Coordinate Gadolinium(III) Complex of 1,7,13-Triaza-4,10, 16-Trioxacyclooctadecane-N,N',N' '-Tri-Acetic Acid. Inorg. Chem. 29, 4366]. Predicted q-values were in complete agreement with experimentally determined q-values. A genetic algorithm-based conformational search method was developed to generate valid 3D models for gadolinium chelates. The method was successfully tested on the following gadolinium chelates: Gd(EDTA) (Templeton et al., 1982), Gd(DOTA) (Dubost et al., 1991), Gd(DTPA-BEA) (Smith et al., 1989), Gd(TTTA) (Chen et al., 1990), Gd(triethylene glycol) [Rogers, R. D., Voss, E. J., and Etzenhouser, R. D. (1988) F-Element Crown Ether Complexes. 17. Synthetic and Structural Survey of Lanthanide Chloride Tiethylene Glycol Complexes. Inorg. Chem. 27, 533], and Gd(tetraethylene glycol) [Rogers, R. D., Etzenhouser, R. D., Murdoch, J. S., and Reyes, E. (1991) Macrocycle Complexation Chemistry. 35. Survey of the Complexation of the Open-Chain 15-Crown-5 Analogue tetraethylene Glycol with the Lanthanide Chlorides. Inorg. Chem. 30, 1445].     

Two-dimensional NMR investigation of iron-sulfur cluster electronic and molecular structure of oxidized Clostridium pasteurianum ferredoxin. Interpretability of contact shifts in terms of cysteine orientation.

A two-dimensional NMR study has been carried out on the four-iron clusters of a bacterial oxidized ferredoxin for the purpose of investigating the relationship between contact shift patterns and the orientation of the individual coordinated cysteines. The ferredoxin from Clostridium pasteurianum, CpFdox, was selected because of its extensive sequence homology, and likely close structural similarity, to the crystallographically characterized ferredoxin from Peptococcus aerogenes, Pa Fdox (Adman, E.T., Sieker, L.C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996). Rapid data collection rates with minimal but adequate acquisition time allowed the detection of numerous CpFdox cross-peaks from the contact-shifted and strongly relaxed coordinated cysteinyl C beta H protons in the resolved 10-20 ppm window. Relatively strong magnitude COSY cross peaks from the resolved eight cysteinyl C beta H resonance unambiguously locate the geminal C beta H partner for each residue; weaker cross-peaks locate the C alpha Hs from three of the residues. The geminal nature of the magnitude-COSY detected partners to the resolved C beta H peaks is confirmed by strong NOESY cross-peaks. The NOESY spectra, moreover, assign an additional two cysteinyl C alpha H resonances. The present results confirm some previous one-dimensional NOE assignments, revise others, and locate resonances previously undetected (Bertini, I., Briganti, F., Luchinat, C., and Scozzafara, A. (1990) Inorg. Chem. 29, 1874-1880). A striking pairwise pseudo-symmetry in cysteinyl contact shift patterns is observed which is attributed to the previously recognized pseudo-symmetry in the crystal of PaFdox. A detailed analysis of the structural/electronic determinants of the coordinated cysteine C beta H contact shift pattern is made, and the NMR data necessary for unique interpretation are identified. It is shown that analysis of the relaxation properties of cysteine beta-methylene protons provides the stereospecific assignments necessary for comparison of shift ratios with crystallographic structural data. The available structural data on PaFdox (Backes, G., Mino, Y., Loehr, T., Meyer, T., Cusanovich, M., Sweeney, W., Adman, E., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 13, 2055-2064) are qualitatively but not quantitatively consistent with the observed cysteinyl contact shift pattern, with the NMR data reflecting more asymmetry than previous studies. A tentative assignment of a single pair of symmetry-related cysteines is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 1. Sequence-specific hydrogen-1 resonance assignments and secondary structure in solution of the oxidized form

Complete sequence-specific assignments were determined for the diamagnetic 1H resonances from Anabaena 7120 ferredoxin (Mr = 11,000). A novel assignment procedure was followed whose first step was the identification of the 13C spin systems of the amino acids by a 13C(13C) double quantum correlation experiment [Oh, B.-H., Westler, M. W., Darba, P., & Markley, J. L. (1988) Science 240, 908-911]. Then, the 1H spin systems of the amino acids were identified from the 13C spin systems by means of direct and relayed 1H(13C) single-bond correlations [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085]. The sequential resonance assignments were based mainly on conventional interresidue 1H alpha i-1HNi + 1 NOE connectivities. Resonances from 18 residues were not resolved in two-dimensional 1H NMR spectra. When these residues were mapped onto the X-ray crystal structure of the homologous ferredoxin from Spirulina platensis [Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., & Matsubara, H. (1980) Nature 286, 522-524], it was found that they correspond to amino acids close to the paramagnetic 2Fe.2S* cluster. Cross peaks in two-dimensional homonuclear 1H NMR spectra were not observed for any protons closer than about 7.8 A to both iron atoms. Secondary structural features identified in solution include two antiparallel beta-sheets, one parallel beta-sheet, and one alpha-helix.     

Studies of the mechanism of phenol hydroxylase: mutants Tyr289Phe, Asp54Asn, and Arg281Met

Three residues in the active site of the flavoprotein phenol hydroxylase (PHHY) were independently changed by site-directed mutagenesis. One of the mutant forms of PHHY, Tyr289Phe, is reduced by NADPH much slower than is the wild-type enzyme, although it has a slightly higher redox potential than the wild-type enzyme. In the structure of the wild-type enzyme, residue Tyr289 is hydrogen-bonded with the FAD when the latter is at the "out" position but has no direct contact with the flavin when it is "in". The oxidative half-reaction of PHHY is not significantly affected by this mutation, contrary to the concept that Tyr289 is a critical residue in the hydroxylation reaction [Enroth, C., Neujahr, H., Schneider, G., and Lindqvist, Y. (1998) Structure 6, 605-617; Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J. (2000) J. Am. Chem. Soc. 122, 8728-8738]. Tyr289 may help stabilize the FAD in the out conformation where it can be reduced by NADPH. For the Asp54Asn mutant form of PHHY, the initial step of the oxidative half-reaction is significantly slower than for the wild-type enzyme. Asp54Asn utilizes less than 20% of the reduced flavin for hydroxylating the substrate with the remainder forming H(2)O(2). Similar changes are observed when Arg281, a residue between Asp54 and the solvent, is mutated to Met. These two residues are suggested to be part of the active site environment the enzyme provides for the flavin cofactor to function optimally in the oxidative half-reaction. In the construction of the mutant forms of PHHY, it was determined that 11 of the previously reported amino acid residues in the sequence of PHHY were incorrect.     

Catalysis-linked inactivation of fluoroacetate dehalogenase by ammonia: a novel approach to probe the active-site environment

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp(105) of the enzyme acts as a nucleophile to attack the alpha-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His(272) [Liu, J.Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated concomitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp(105), into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for characterizing the active-site environment as well as for identifying the catalytic residue.     

The interaction of acetate and formate with cobalt carbonic anhydrase. An NMR study.

The interaction of formate and acetate ions with cobalt-substituted carbonic anhydrase (CA) has been investigated through 13C-NMR and one-dimensional and two-dimensional 1H-NMR spectroscopy. 13C data on formate are consistent with a regularly coordinated ligand, as previously proposed for the acetate anion [Bertini, I., Luchinat, C. & Scozzafava, A. (1977) J. Chem. Soc. Dalton Trans., 1962-1965]. 1H-NOE experiments on both anions give evidence of through-space interactions between ligand protons and protein protons. The latter are assigned to specific residues in the active cavity through nuclear Overhauser effect spectroscopy (NOESY) experiments. The 13C-derived and 1H-derived constrains allow reliable docking of these ligands in the active-site cavity. The resulting geometries are similar to one another and consistent with five-coordinated structures around the metal ion, as previously proposed from electronic spectroscopy [Bertini, I., Canti, G., Luchinat, C. & Scozzafava, A. (1978) J. Am. Chem. Soc. 100, 4873-4877]. The results are discussed in light of the current debate on anion binding to metal ions in carbonic anhydrase [Lindahl, M., Svensson, A. & Liljas, A. (1992) Proteins, in the press]; Bertini, I., Luchinat, C., Pierattelli, R. & Vila, A. J. (1992) Inorg. Chem., in the press; Banci, L. & Merz, K. (1992) unpublished results] and, in particular, of the proposed long Zn-O distance found in the recent X-ray results on the formate adduct [Hakanson, K., Carlsson, M., Svensson, A. & Liljas, A. (1992) J. Mol. Biol., in the press].     

Rapid and quantitative activation of Chlamydia trachomatis ribonucleotide reductase by hydrogen peroxide

We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor in its R2 subunit to initiate catalysis [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The Mn (IV) site of the novel cofactor functionally replaces the tyrosyl radical used by conventional class I RNRs to initiate substrate radical production. As a first step in evaluating the hypothesis that the use of the alternative cofactor could make the RNR more robust to reactive oxygen and nitrogen species [RO(N)S] produced by the host's immune system [H?gbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gr?slund, A., and Nordlund, P. (2004) Science 305, 245-248], we have examined the reactivities of three stable redox states of the Mn/Fe cluster (Mn (II)/Fe (II), Mn (III)/Fe (III), and Mn (IV)/Fe (III)) toward hydrogen peroxide. Not only is the activity of the Mn (IV)/Fe (III)-R2 intermediate stable to prolonged (>1 h) incubations with as much as 5 mM H 2O 2, but both the fully reduced (Mn (II)/Fe (II)) and one-electron-reduced (Mn (III)/Fe (III)) forms of the protein are also efficiently activated by H 2O 2. The Mn (III)/Fe (III)-R2 species reacts with a second-order rate constant of 8 +/- 1 M (-1) s (-1) to yield the Mn (IV)/Fe (IV)-R2 intermediate previously observed in the reaction of Mn (II)/Fe (II)-R2 with O 2 [Jiang, W., Hoffart, L. M., Krebs, C., and Bollinger, J. M., Jr. (2007) Biochemistry 46, 8709-8716]. As previously observed, the intermediate decays by reduction of the Fe site to the active Mn (IV)/Fe (III)-R2 complex. The reaction of the Mn (II)/Fe (II)-R2 species with H 2O 2 proceeds in three resolved steps: sequential oxidation to Mn (III)/Fe (III)-R2 ( k = 1.7 +/- 0.3 mM (-1) s (-1)) and Mn (IV)/Fe (IV)-R2, followed by decay of the intermediate to the active Mn (IV)/Fe (III)-R2 product. The efficient reaction of both reduced forms with H 2O 2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe 2 (II/II)) to the "met" (Fe 2 (III/III)) form [Gerez, C., and Fontecave, M. (1992) Biochemistry 31, 780-786] but is then only very inefficiently converted from the met to the active (Fe 2 (III/III)-Y (*)) form [Sahlin, M., Sj?berg, B.-M., Backes, G., Loehr, T., and Sanders-Loehr, J. (1990) Biochem. Biophys. Res. Commun. 167, 813-818].     

A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL

Apoptosis-inducing ligand 2 (Apo2L, also called TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in a variety of human tumor cell lines but not in normal cells [Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673-682; Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687-12690]. Here we describe the structure of Apo2L at 1.3 A resolution and use alanine-scanning mutagenesis to map the receptor contact regions. The structure reveals a homotrimeric protein that resembles TNF with receptor-binding epitopes at the interface between monomers. A zinc ion is buried at the trimer interface, coordinated by the single cysteine residue of each monomer. The zinc ion is required for maintaining the native structure and stability and, hence, the biological activity of Apo2L. This is the first example of metal-dependent oligomerization and function of a cytokine.     

Direct photoaffinity labeling of gizzard myosin with [3H]uridine diphosphate places Glu185 of the heavy chain at the active site

The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with [3H]UDP. [3H] UDP was stably trapped at the active site by addition of vanadate (Vi) and Co2+. The extraordinary stability of the myosin.Co2+.[3H]UDP.Vi complex (t1/2 greater than 5 days at 0 degrees C) allowed it to be purified free of extraneous [3H]UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped [3H]UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results (Maruta, H., and Korn, E. (1981) J. Biol. Chem. 256, 499-502) using [3H]UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a "zero-length" cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by [3H]UTP photolabeling in Acanthamoeba myosin II (Atkinson, M. A., Robinson, E. A., Appella, E., and Korn, E. D. (1986) J. Biol. Chem. 261, 1844-1848).     

Mediation by indole analogues of electron transfer during oxygen activation in variants of Escherichia coli ribonucleotide reductase R2 lacking the electron-shuttling tryptophan 48

Activation of dioxygen by the carboxylate-bridged diiron(II) cluster in the R2 subunit of class I ribonucleotide reductase from Escherichia coli results in the one-electron oxidation of tyrosine 122 (Y122) to a stable radical (Y122*). A key step in this reaction is the rapid transfer of a single electron from a near-surface residue, tryptophan 48 (W48), to an adduct between O(2) and diiron(II) cluster to generate a readily reducible cation radical (W48(+)(*)) and the formally Fe(IV)Fe(III) intermediate known as cluster X. Previous work showed that this electron injection step is blocked in the R2 variant with W48 replaced by phenylalanine [Krebs, C., Chen, S., Baldwin, J., Ley, B. A., Patel, U., Edmondson, D. E., Huynh, B. H., and Bollinger, J. M., Jr. (2000) J. Am. Chem. Soc. 122, 12207-12219]. In this study, we show that substitution of W48 with alanine similarly disables the electron transfer (ET) but also permits its chemical mediation by indole compounds. In the presence of an indole mediator, O(2) activation in the R2-W48A variant produces approximately 1 equiv of stable Y122* and more than 1 equiv of the normal (micro-oxo)diiron(III) product. In the absence of a mediator, the variant protein generates primarily altered Fe(III) products and only one-fourth as much stable Y122* because, as previously reported for R2-W48F, most of the Y122* that is produced decays as a consequence of the inability of the protein to mediate reductive quenching of one of the two oxidizing equivalents of the initial diiron(II)-O(2) complex. Mediation of ET is effective in W48A variants containing additional substitutions that also impact the reaction mechanism or outcome. In the reaction of R2-W48A/F208Y, the presence of mediator suppresses formation of the Y208-derived diiron(III)-catecholate product (which is predominant in R2-F208Y in the absence of reductants) in favor of Y122*. In the reaction of R2-W48A/D84E, the presence of mediator affects the outcome of decay of the peroxodiiron(III) intermediate known to accumulate in D84E variants, increasing the yield of Y122* by as much as 2.2-fold to a final value of 0.75 equiv and suppressing formation of a 490 nm absorbing product that results from decay of the two-electron oxidized intermediate in the absence of a functional ET apparatus.     

Evidence for a redox-linked ionizable group associated with the [2Fe-2S] cluster of Thermus Rieske protein

The [2Fe-2S] clusters of Thermus Rieske protein, which were previously found to have nitrogen atoms coordinated directly to the iron (Cline, J.F., Hoffman, B.M., LaHaie, E., Ballou, D.P., and Fee, J.A. (1985) J. Biol. Chem. 260, 3251-3254), are now shown to have a tightly linked ionization that affects the spectral and redox properties of the cluster. The data are consistent with the reactions LH+, Fe3+ in equilibrium with L-Fe3+ +H+ and L-Fe3+ + H+ + e in equilibrium with LH+, Fe2+, where L is coordinated to Fe3+ but LH+ may not be, depending on its structure. The pKa of the protonic equilibrium is approximately 8 and the midpoint potential, Em7, is approximately 140 mV. Possible structures of L are suggested.