A single methionine residue dictates the kinetic mechanism of interprotein electron transfer from methylamine dehydrogenase to amicyanin

Amicyanin is a type 1 copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). A P52G amicyanin mutation increased the Kd for complex formation and caused the normally true electron transfer (ET) reaction from O-quinol MADH to amicyanin to become a gated ET reaction (Ma, J. K., Carrell, C. J., Mathews, F. S., and Davidson, V. L. (2006) Biochemistry 45, 8284-8293). One consequence of the P52G mutation was to reposition the side chain of Met51, which is present at the MADH-amicyanin interface. To examine the precise role of Met51 in this interprotein ET reaction, Met51 was converted to Ala, Lys, and Leu. The Kd for complex formation of M51A amicyanin was unchanged but the experimentally determined electronic coupling increased from 12 cm-1 to 142 cm-1, and the reorganization energy increased from 2.3 to 3.1 eV. The rate and salt dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin is also changed by the M51A mutation. These changes in ET parameters and rates for the reactions with M51A amicyanin were similar to those caused by the P52G mutation and indicated that the ET reaction had become gated by a similar process, most likely a conformational rearrangement of the protein ET complex. The results of the M51K and M51L mutations also have consequences on the kinetic mechanism of regulation of the interprotein ET with effects that are intermediate between what is observed for the reaction of the native amicyanin and M51A amicyanin. These data indicate that the loss of the interactions involving Pro52 were primarily responsible for the change in Kd for P52G amicyanin, while the interactions involving the Met51 side chain are entirely responsible for the change in ET parameters and conversion of the true ET reaction of native amicyanin into a conformationally gated ET reaction.     

The sequence of a peptide containing the active site phosphohistidine residue of phosphoglycerate mutase from chicken breast muscle.

Phosphoglycerate mutase is phosphorylated on a histidine residue by the cofactor of the reaction, 2,3-bisphosphoglycerate (Rose, Z. B. (1970) Arch. Biochem. Biophys. 140, 508-513). The phosphoryl group is readily transferred to the normal acceptors, 3-phosphoglycerate and 2-phosphoglycerate, or to water in the presence of glycolate-2-P. An acid-labile phosphorylated decapeptide has been purified from a tryptic digest of the phosphoenzyme. The amino acid sequence of the peptide has been determined to be: Aal-Gly-Gln-Leu-Asp-Glu-Ser-His-Arg. This sequence bears a striking analogy to part of a highly conserved region of lactate dehydrogenase (residues 100 to 109) (Taylor, S. S., Oxley, S. S., Allison, W. S., and Kaplan, N. O. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1970-1974). Evidence from x-ray crystallographic studies indicates that the two enzymes are similar in tertiary structure (Campbell, J. W., Watson, H. C. and Hodgson, G. I. (1974) Nature 250, 301-303).     

Active site serine promotes stabilization of the reactive glutathione thiolate in rat glutathione transferase T2-2. Evidence against proposed sulfatase activity of the corresponding human enzyme

Ser(11) in rat glutathione transferase T2-2 is important for stabilization of the reactive enzyme-bound glutathione thiolate in the reaction with 1-menaphthyl sulfate. The S11A mutation increased the pK(a) value for the pH dependence of the rate constant for pre-steady-state product formation, from 5.7 to 7.9. This pH dependence is proposed to reflect titration of enzyme-bound glutathione thiol. Further, the mutation lowered the k(cat) value but not because of the impaired stabilization of the glutathione thiolate. In fact, several steps on the reaction pathway were affected by the S11A mutation, and the cause of the decreased k(cat) for the mutant was found to be a slower product release. The data presented here contradict the hypothesis that glutathione transferase T2-2 could act as a sulfatase that is not dependent on Ser(11) for the catalytic activity, as proposed for the corresponding human enzyme (Tan, K.-L., Chelvanayagam, G., Parker, M. W., and Board, P. G. (1996) Biochem. J. 319, 315-321; Rossjohn, J., McKinstry, W. J., Oakley, A. J., Verger, D., Flanagan, J., Chelvanayagam, G., Tan, K.-L., Board, P. G., and Parker, M. W. (1998) Structure 6, 309-322). On the contrary, Ser(11) governs both chemical and physical steps of the catalyzed reaction.     

Characterization of the tryptophan-derived quinone cofactor of methylamine dehydrogenase by resonance Raman spectroscopy

The resonance Raman (RR) spectrum of oxidized methylamine dehydrogenase (MADHOX) exhibits a set of C-H, C-C, C = C, and C = O vibrational modes between 900 and 1700 cm-1 that are characteristic of the quinone moiety of the tryptophan tryptophlyquinone (TTQ) cofactor. The close similarity of the RR spectra for MADHs from Paracoccus denitrificans (Pd), Thiobacillus versutus (Tv), and bacterium W3A1 proves that the same cofactor is present in all three proteins. The MADHs from Pd and Tv have a v(C = O) mode at approximately 1625 cm-1 that shifts approximately 20 cm-1 upon 18O substitution of one of the carbonyl oxygens and is assigned to the in-phase symmetric stretch of the two C = O groups. The semiquinone form of Pd MADH has its own characteristic RR spectrum with altered peak frequencies and intensities as well as a decrease in the total number of peaks. The hydroxide and ammonia adducts of MADHOX produce RR spectra similar to that of the semiquinone. The spectral changes in all three cases are interpreted as being due to reduced conjugation of the cofactor. The ammonia adduct is formulated as a carbinolamine, a likely intermediate in the enzymatic mechanism. In contrast, formation of the electron-transfer complex between amicyanin and MADHOX has no effect on the vibrational frequencies (and, hence, structure) of either the MADH quinone or the amicyanin blue copper site. The behavior of the TTQ cofactors of Pd and Tv MADHs are very similar to one another and somewhat different from W3A1 MADH, particularly with regard to adduct formation and ability to undergo isotope exchange with solvent. These differences are ascribed to the cofactor environments within the proteins rather than to the structure of the cofactor itself.     

Characterization of the regulatory thioredoxin site of phosphoribulokinase

Phosphoribulokinase is light-regulated via thioredoxin by reversible oxidation/reduction of sulfhydryl/disulfide groups. To identify the cysteinyl residues that are involved in regulation, the S-carboxymethyl labeling patterns of the fully reduced (active) and oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced, [14C]carboxymethylated enzyme contained four labeled peptides, all of which were purified and sequenced by Edman degradation. If the enzyme was oxidized by 5,5'-dithiobis-(2-nitrobenzoic acid) prior to carboxymethylation and tryptic digestion, only two labeled peptides were observed, thereby revealing the identity of the regulatory cysteines as Cys-16 and Cys-55. The former was previously implicated as part of the nucleotide-binding domain of the active site (Porter, M.A., and Hartman, F.C. (1986) Biochemistry 25, 7314-7318), a conclusion reinforced by the present observation that the sequence around the Cys-16 is similar to a consensus sequence of ATP-binding sites from a number of proteins of diverse phylogenetic origin (Higgins, C.F., Hiles, I.D., Salmond, G.P.C., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D., Bell, A.W., and Hermondson, M. (1986) Nature 323, 448-450). The regulatory disulfide of phosphoribulokinase was found to be intrasubunit based on the stoichiometry of the oxidation and the failure to resolve oxidized and reduced enzyme by gel filtration under dissociation conditions.     

A 1H-NMR study of electronic structure of the active site of Galeorhinus japonicus metmyoglobin

The ferric high-spin form of the myoglobin from the shark Galeorhinus japonicus, which possesses a Gln residue at the distal site instead of the usual His residue, has been studied by 1H-NMR spectroscopy. Using the heme meso-proton (C5H, C10H, C15H and C20H) resonance shift as a diagnostic probe for identifying the coordination system of the iron center in ferric high-spin form of hemoprotein, it has been shown that G. japonicus metmyoglobin (metMb) possesses the pentacoordinated active site. The pH-dependence study of NMR spectra of G. japonicus metMb revealed the appearance of the hydroxyl form of metMb at high pH, indicating that the protein undergoes the transition between the acidic and alkaline forms. The pK value and the rate for this acid-alkaline transition in G. japonicus metMb were found to be approximately 10 and much less than 4 x 10(2) s-1, respectively. Since the pK value of the acid-alkaline transition for the pentacoordinated heme in Aplysia limacina metMb is 7.8 [Giacometti, G.M., Das Ros, A., Antonini, E. & Brunori, M. (1975) Biochemistry 14, 1584-1588] and that of the hexacoordinated heme in sperm whale metMb is 9.1 [Brunori, M., Antonini, E., Fasella, P., Wyman, J. & Rossi-Fanelli, A. (1968) J. Mol. Biol. 34, 497-504], the OH- affinity of the ferric heme iron does not appear to depend on its coordination system. The acid-alkaline transition rate in A. limacina metMb was reported to be much less than 1.5 x 10(2) s-1 [Pande, U., La Mar, G.N., Lecomte, J.T.J., Ascoli, F., Brunori, M., Smith, K.M., Pandey, R.K., Parish, D.W. & Thanabal, V. (1986) Biochemistry 25, 5638-5646] and therefore a slow transition rate may be unique to the pentacoordinated active site of Mb.     

Probes of a role for remote binding interactions on hydrogen tunneling in the horse liver alcohol dehydrogenase reaction

A tunneling contribution to hydride transfer has been demonstrated previously in the oxidation of benzyl alcohol catalyzed by an active-site mutant (F93W) of horse liver alcohol dehydrogenase (LADH) [Bahnson, B. J., et al. (1993) Biochemistry 32, 5503-5507]. Mutation of a residue that lies directly behind the nicotinamide ring of the bound cofactor has further shown that side-chain bulk can contribute to catalytic efficiency and tunneling in a correlated fashion [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. Second site mutations of F93W have now been made at positions more remote from the active site. In particular, we have focused on an isoleucine residue that interacts with the adenine moiety of the NAD(+) cofactor, 20 A from the nicotinamide ring. Replacement of this remote residue with glycine (F93W:I224G), alanine (F93W:I224A), valine (F93W:I224V), and leucine (F93W:I224L) is concluded to destabilize the binding of NAD(+). All double mutants exhibited a K(M) for NAD(+) that is 2-25 times higher than that for the F93W enzyme. However, neither the catalytic efficiency for turnover of benzyl alcohol [k(cat)/K(M(benzyl alcohol))] nor the relationship between the secondary k(H)/k(T) and k(D)/k(T) isotope effects for benzyl alcohol oxidation was significantly affected. The lack of differences observed in the isotope effects indicates that these mutations have little effect on the extent of hydrogen tunneling in the reaction. The complete removal of the side chain at position 224 in the F93W:I224G enzyme resulted in a less than 5% decrease in the ratio of the secondary isotope effects, maintaining the ratio above the semiclassical limit for the indication of tunneling in the reaction. By contrast, K(i) for NAD(+) increased 60-fold for this mutant. The results obtained with F93W:I224G are consistent with remote interactions that affect the association and binding of cofactor in a reactive conformation. However, once this conformation is achieved, hydride transfer and its tunneling component proceed as with the single F93W mutant enzyme, uninfluenced by the remote mutation. Replacement of other side chains, with alpha-carbon positions from about 8 to over 20 A from the C4 position of the nicotinamide ring, demonstrated a similar insensitivity of k(cat)/K(M(benzyl alcohol)) to protein modification. Comparison to earlier studies with active-site mutants of LADH implicates a role for proximal, but not distal, side chains in the modulation of hydrogen tunneling for this enzyme.     

Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access

Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.     

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].     

Structural evidence for leucine at the reactive site of heparin cofactor II

The reaction products formed during the enzymatic inactivation of heparin cofactor II (HCII) by a proteinase isolated from Echis carinatus were analyzed by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis and by reverse-phase high-performance liquid chromatography. By NaDodSO4-polyacrylamide gel electrophoresis, limited proteolysis of HCII was observed, which resulted in a decrease in the apparent molecular weight of the protein from approximately 68 000 to approximately 53 000. By reverse-phase high-performance liquid chromatography, at least 20 peptides were observed. Primary structure analysis of these peptides indicated that significant proteolysis had occurred in the NH2-terminal region of the protein. HCII inactivation, however, coincided with the appearance of a peptide from the COOH-terminal region of the protein. The peptide differed from the previously identified reactive site peptide [Griffith, M. J., Noyes, C. M., & Church, F. C. (1985) J. Biol. Chem. 260, 2218-2225] by only one residue: a leucyl residue at the NH2-terminal of the peptide. We conclude that leucine, as opposed to the expected arginine, is at the reactive site of HCII.     

Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.     

Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

Previous amino acid substitutions at the M4 domain of the Torpedo californica and mouse acetylcholine receptor suggested that the location of the substitution relative to the membrane-lipid interface and perhaps to the ion pore can be critical to the channel gating mechanism [Lasalde, J. A., Tamamizu, S., Butler, D. H., Vibat, C. R. T., Hung, B., and McNamee, M. G. (1996) Biochemistry 35, 14139-14148; Ortiz-Miranda, S. I., Lasalde, J. A., Pappone, P. A., and McNamee, M. G. (1997) J. Membr. Biol. 158, 17-30; Tamamizu, S., Lee, Y. H., Hung, B., McNamee, M. G., and Lasalde-Dominicci, J. A. (1999) J. Membr. Biol. 170, 157-164]. In this study, we introduce tryptophan substitutions at 12 positions (C412W, M415W, L416W, I417W, C418W, I419W, I420W, G421W, T422W, V423W, S424W, and V425W) along this postulated lipid-exposed segment M4 so that we can examine functional consequences on channel gating. The expression levels of mutants C412W, G421W, S424W, and V425W were almost the same as that of the wild type, whereas other mutants (M415W, L416W, C418W, I419W, I420W, T422W, and V423W) had relatively lower expression levels compared to that of the wild type as measured by iodinated alpha-bungarotoxin binding ([(125)I]-alpha-BgTx). Two positions (L416W and I419W) had less than 20% of the wild type expression level. I417W gave no detectable [(125)I]BgTx binding on the surface of oocyte, suggesting that this position might be involved in the AChR assembly, oligomerization, or transport to the cell membrane. The alphaV425W mutant exhibited a significant increase in the open channel probability with a moderate increase in the macroscopic response at higher ACh concentrations very likely due to channel block. The periodicity for the alteration of receptor assembly and ion channel function seems to favor a potential alpha-helical structure. Mutants that have lower levels of expression are clustered on one side of the postulated alpha-helical structure. Mutations that display normal expression and functional activity have been shown previously to face the membrane lipids by independent labeling studies. The functional analysis of these mutations will be presented and discussed in terms of possible structural models.     

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.     

Re-engineering monovalent cation binding sites of methylamine dehydrogenase: effects on spectral properties and gated electron transfer

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-dependent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from substrate-reduced MADH to amicyanin. Two putative monovalent cation binding sites in MADH have been identified by X-ray crystallography [Labesse, G., Ferrari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathews, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi interactions involving residue alpha Phe55. An alpha F55A mutation differentially affects these two monovalent cation-dependent phenomena. The apparent K(d) associated with spectral perturbations increases 10-fold. The apparent K(d) associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the requirement for the cation for the gated ET. These results show that of the two binding sites revealed in the structure, cation binding to the distal site, which is stabilized by the cation-pi interactions, is responsible for the spectral perturbations. Cation binding to the proximal site, which is stabilized by several oxygen ligands, is responsible for the enhancement of the rate of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited cation binding properties that were the same as those of the native enzyme, indicating that interactions with the carboxylate of Glu can effectively replace the cation-pi interactions with Phe in stabilizing monovalent cation binding to the distal site.     

Identification of a uniquely immunodominant, cross-reacting site in the human immunodeficiency virus endonuclease protein.

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.     

Ubiquinone binding, ubiquinone exclusion, and detailed cofactor conformation in a mutant bacterial reaction center

The X-ray crystal structure of a Rhodobacter sphaeroides reaction center with the mutation Ala M260 to Trp (AM260W) has been determined. Diffraction data were collected that were 97.6% complete between 30.0 and 2.1 A resolution. The electron density maps confirm the conclusions of a previous spectroscopic study, that the Q(A) ubiquinone is absent from the AM260W reaction center (Ridge, J. P., van Brederode, M. E., Goodwin, M. G., van Grondelle, R., and Jones, M. R. (1999) Photosynthesis Res. 59, 9-26). Exclusion of the Q(A) ubiquinone caused by the AM260W mutation is accompanied by a change in the packing of amino acids in the vicinity of the Q(A) site that form part of a loop that connects the DE and E helices of the M subunit. This repacking minimizes the volume of the cavity that results from the exclusion of the Q(A) ubiquinone, and further space is taken up by a feature in the electron density maps that has been modeled as a chloride ion. An unexpected finding is that the occupancy of the Q(B) site by ubiquinone appears to be high in the AM260W crystals, and as a result the position of the Q(B) ubiquinone is well-defined. The high quality of the electron density maps also reveals more precise information on the detailed conformation of the reaction center carotenoid, and we discuss the possibility of a bonding interaction between the methoxy group of the carotenoid and residue Trp M75. The conformation of the 2-acetyl carbonyl group in each of the reaction center bacteriochlorins is also discussed.     

AurF from Streptomyces thioluteus and a possible new family of manganese/iron oxygenases

We recently reported that the R2 subunit of class Ic ribonucleotide reductase from Chlamydia trachomatis contains a heterodinuclear Mn/Fe redox cofactor [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 N-oxygenase, AurF, from Streptomyces thioluteus catalyzes the six-electron oxidation of p-aminobenzoate to p-nitrobenzoate and contains the EX2HX60-180EX2H sequence motif previously used to identify proteins with non-heme diiron clusters. Two research groups independently obtained evidence for the presence of iron and manganese in preparations of AurF. The electron paramagnetic resonance (EPR) spectrum of purified, resting AurF presented in one of these studies is markedly similar to the spectrum of the MnIII/FeIII form of C. trachomatis R2. We propose that S. thioluteus AurF also may harbor a heterodinuclear Mn/Fe cofactor, which it may use to activate O2 for oxidation of the aryl amine to the nitro compound. Hypothetical proteins encoded in the genomes of several other bacteria have similar sequences and may also be members of this nascent family of oxygen-activating Mn/Fe proteins.     

A manganese(IV)/iron(IV) intermediate in assembly of the manganese(IV)/iron(III) cofactor of Chlamydia trachomatis ribonucleotide reductase

We recently showed that the class Ic ribonucleotide reductase from the human pathogen Chlamydia trachomatis uses a Mn(IV)/Fe(III) cofactor to generate protein and substrate radicals in its catalytic mechanism [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]. Here, we have dissected the mechanism of formation of this novel heterobinuclear redox cofactor from the Mn(II)/Fe(II) cluster and O2. An intermediate with a g = 2 EPR signal that shows hyperfine coupling to both 55Mn and 57Fe accumulates almost quantitatively in a second-order reaction between O2 and the reduced R2 complex. The otherwise slow decay of the intermediate to the active Mn(IV)/Fe(III)-R2 complex is accelerated by the presence of the one-electron reductant, ascorbate, implying that the intermediate is more oxidized than Mn(IV)/Fe(III). M?ssbauer spectra show that the intermediate contains a high-spin Fe(IV) center. Its chemical and spectroscopic properties establish that the intermediate is a Mn(IV)/Fe(IV)-R2 complex with an S = 1/2 electronic ground state arising from antiferromagnetic coupling between the Mn(IV) (S(Mn) = 3/2) and high-spin Fe(IV) (S(Fe) = 2) sites.     

Structure-activity studies of the inhibition of FabI,the enoyl reductase from Escherichia coli,by triclosan: kinetic analysis of mutant FabIs

Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type II bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E.NAD(+) form of the wild-type enzyme with a K(1) value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K(1) values of 0.2 microM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K(2) value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3 and 30 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.