Methionine-rich motifs have an important role in copper trafficking factors, including the CusF protein. Here we show that CusF uses a new metal recognition site wherein Cu(I) is tetragonally displaced from a Met 2 His ligand plane toward a conserved tryptophan. Spectroscopic studies demonstrate that both thioether ligation and strong cation-π interactions with tryptophan stabilize metal binding. This novel active site chemistry affords mechanisms for control of adventitious metal redox and substitution chemistry.In recent years, metal-specific gene regulatory and cation-trafficking proteins have been isolated and demonstrate metal binding motifs with unprecedented coordination chemistry tailored to their function 1 . For example, the CXXC sequence, found in cytosolic copper chaperones and trafficking proteins, provides for facile Cu(I) transfer via low-coordinationnumber anionic intermediates 1,2 . Extracellular or periplasmic copper trafficking domains, however, function in environments that are more oxidizing than the cytosol and frequently have less well understood methionine-rich sequences 3-8 . The cus operon encodes a bacterial copper homeostasis system with several methionine-motif proteins 5,9,10 , including the periplasmic protein CusF, which is thought to serve as copper chaperone or regulator 5,6 . CusF binds Cu(I) in vitro 11 , and a methionine-rich Cu(I) site was proposed 6 based on an apo-CusF structure and NMR chemical shift data. Here we show that metal recognition in CusF involves a strong interaction between a cationic Cu(I)-thioether/imidazole center and the aromatic ring of tryptophan. To our knowledge, such cation-π interactions have not been reported for transition metal receptors or metalloenzyme active sites.Correspondence should be addressed to T.V.O. (t-ohalloran@northwestern.edu). 6 These authors contributed equally to this work.Published online at http://www.nature.com/naturechemicalbiology Reprints and permissions information is available online at

Raman spectra of proteins that are obtained with deep ultraviolet excitation contain resonance-enhanced amide bands of the polypeptide backbone, as well as aromatic side chain bands. The amide bands are sensitive to conformation, and can be used to estimate the backbone secondary structure. UV Raman spectra are reported at 206.5 and 197 nm, for a set of 12 proteins with varied secondary structure content, and are used to establish quantitative signatures of secondary structure via least-squares fitting. Amide band enhancement is greater at 197 nm, where basis spectra are established for b-turn, as well as a-helix, b-sheet and unordered structures; the lower signal strength at 206.5 nm does not provide a reliable spectrum for the first of these. Application of these basis spectra is illustrated for the melting of apo-myoglobin. The amide band positions and cross sections are discussed.

The gaseous XO molecules (X = C, N or O) bind to the heme prosthetic group of heme proteins, and thereby activate or inhibit key biological processes. These events depend on interactions of the surrounding protein with the FeXO adduct, interactions that can be monitored via the frequencies of the Fe-X and X-O bond stretching modes, νFeX and νXO. The frequencies can be determined by vibrational spectroscopy, especially resonance Raman spectroscopy. Backbonding, the donation of Fe dπ electrons to the XO π* orbitals, is a major bonding feature in all the FeXO adducts. Variations in backbonding produce negative νFeX/νXO correlations, which can be used to gauge electrostatic and H-bonding effects in the protein binding pocket. Backbonding correlations have been established for all the FeXO adducts, using porphyrins with electron donating and withdrawing substituents. However the adducts differ in their response to variations in the nature of the axial ligand, and to specific distal interactions. These variations provide differing vantages for evaluating the nature of protein-heme interactions. We review experimental studies that explore these variations, and DFT computational studies that illuminate the underlying physical mechanisms.

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