Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH approximately 6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.

The multicopper oxidases contain at least four copper atoms and catalyze the four-electron reduction of O(2) to H(2)O at a trinuclear copper cluster. An intermediate, termed native intermediate, has been trapped by a rapid freeze-quench technique from Rhus vernicifera laccase when the fully reduced form reacts with dioxygen. This intermediate had been described as an oxygen-radical bound to the trinuclear copper cluster with one Cu site reduced. XAS, however, shows that all copper atoms are oxidized in this intermediate. A combination of EXAFS, multifrequency EPR, and VTVH MCD has been used to understand how this fully oxidized trinuclear Cu cluster relates to the fully oxidized resting form of the enzyme. It is determined that in the native intermediate all copper atoms of the cluster are bridged by the product of full O(2) reduction. In contrast, the resting form has one copper atom of the cluster (the T2 Cu) magnetically isolated from the others. The native intermediate decays to the resting oxidized form with a rate that is too slow to be in the catalytic cycle. Thus, the native intermediate appears to be the catalytically relevant fully oxidized form of the enzyme, and its role in catalysis is considered.

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92-96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin-echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.Prion diseases are fatal neurodegenerative disorders of both humans and animals (1). The causative agent is an isoform of a normal, host-encoded membrane glycoprotein called the prion protein (PrP). 1 The normal cellular isoform (PrP C ) is the precursor to the pathogenic, protease-resistant isoform termed PrP Sc , which is responsible for homologous pathologies within its individual hosts. With rare but notable exceptions, prion diseases respect the species barrier (2,3). Among these exceptions is the transmission of disease from scrapie-infected † This work was supported by NIH Grants GM 65790 (G.L.M.), GM 60609 (G.J.G.), GM 40168 (J.P.), AG02132 and AG10770 (S.B.P.).

NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page.

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 . Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 M) to 95 M. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/ protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.Methanotrophs are a group of gram-negative bacteria that utilize methane or methanol as the sole source of carbon and energy (1,20). The initial oxidation of methane to methanol is catalyzed by methane monooxygenase (MMO). In some methanotrophs, two different MMOs can be expressed, depending on the copper concentration during growth (11, 37, 39): a soluble cytoplasmic MMO (sMMO) and a membrane-associated, or particulate, MMO (pMMO). In cells cultured under low copper/biomass ratios (Յ0.9 nmol of Cu/mg of cell protein), the sMMO is expressed (20, 28). Cells cultured under higher copper/biomass ratios express pMMO, and there is no detectable sMMO expression (35,43). While sMMO is a wellcharacterized enzyme that consists of a hydroxylase component composed of three polypeptides and a hydroxo-bridged binuclear iron cluster-an NADH-dependent reductase component composed of one polypeptide containing both FAD and [Fe 2 S 2 ] cofactors and a regulatory polypeptide (18,26,27,31,47)-information on the molecular properties of pMMO is limited due to the instability of pMMO in cell-free fractions.Purification of the pMMO has been reported from Methylococcus capsulatus Bath (2,25,33, 52) and M. trichosporium

The intermediate, designated X, formed during the self-assembly reaction of the tyrosyl radical/µoxo-bridged diferric cofactor in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) is directly involved in the oxidation of Y122 to the catalytically essential ‚Y122. Earlier rapid freeze-quench (RFQ) Q-band ENDOR studies led to the formulation of X as a spin-coupled Fe III /Fe IV center, with an S ) 1 / 2 ground state, and showed that X contains a single terminal aqua ligand (water molecule or 2-fold disordered hydroxyl) bound to Fe III but does not contain an hydroxyl bridge. That ENDOR data, coupled with RFQ-EXAFS data, plus the strong spin coupling between the iron ions constrain the structure of X to a di-or tribridged species whose inorganic core (defined as iron and exogenous ligands) contains the [(H x O)Fe III OFe IV ] fragment. To determine whether the core contains a second oxo bridge and to establish the fate of the atoms derived from O 2 , we have now performed CW and pulsed Q-band 17 O ENDOR experiments on samples of X prepared in both H 2 17 O and 17 O 2 , using a uniformly 15 N-labeled protein, [U-15 N]-R2. These measurements, along with kinetic studies on the formation of X in both wild-type and Y122F R2, as monitored by both ENDOR and S-band EPR spectroscopies, reveal that X contains two oxygen atoms. Both are initially derived from O 2 , with one present as a µ-oxo bridge and one as the terminal aqua ligand; with time the latter of these atoms exchanges with solvent. These and our previous studies indicate that X does not contain a di-µ-oxo-or µ-oxo,hydroxobridged core structure. A structure for X is proposed that contains a single oxo bridge, one terminal aqua ligand bound to the Fe III , and one or two additional mono-oxo bridges provided by the carboxylate oxygens of E115 and/or E238. In addition, the time course of the formation of X in the presence of 17 O 2 provides important insights into the dynamics of cluster assembly.

To examine the potential role of methanobactin (mb) as the extracellular component of a copper acquisition system in Methylosinus trichosporium OB3b, the metal binding properties of mb were examined. Spectral (UV-visible, fluorescence, and circular dichroism), kinetic, and thermodynamic data suggested copper coordination changes at different Cu(II):mb ratios. Mb appeared to initially bind Cu(II) as a homodimer with a comparatively high copper affinity at Cu(II):mb ratios below 0.2, with a binding constant (K) greater than that of EDTA (log K = 18.8) and an approximate DeltaG degrees of -47 kcal/mol. At Cu(II):mb ratios between 0.2 and 0.45, the K dropped to (2.6 +/- 0.46) x 10(8) with a DeltaG degrees of -11.46 kcal/mol followed by another K of (1.40 +/- 0.21) x 10(6) and a DeltaG degrees of -8.38 kcal/mol at Cu(II):mb ratios of 0.45-0.85. The kinetic and spectral changes also suggested Cu(II) was initially coordinated to the 4-thiocarbonyl-5-hydroxy imidazolate (THI) and possibly Tyr, followed by reduction to Cu(I), and then coordination of Cu(I) to 4-hydroxy-5-thiocarbonyl imidazolate (HTI) resulting in the final coordination of Cu(I) by THI and HTI. The rate constant (k(obsI)) of binding of Cu(II) to THI exceeded that of the stopped flow apparatus that was used, i.e., >640 s(-)(1), whereas the coordination of copper to HTI showed a 6-8 ms lag time followed by a k(obsII) of 121 +/- 9 s(-)(1). Mb also solubilized and bound Cu(I) with a k(obsI) to THI of >640 s(-)(1), but with a slower rate constant to HTI (k(obsII) = 8.27 +/- 0.16 s(-)(1)), and appeared to initially bind Cu(I) as a monomer.