Low-temperature absorption and room temperature circular dichroism and magnetic circular dichroism spectral studies of the blue copper proteins Rhus vernicifera stellacyanin, bean plastocyanin, and Pseudomonas aeruginosa azurin have been made. Low-energy bands attributable to d-d transitions in a flattened tetrahedral (£>2

Distorted tetrahedral Cu(II) should exhibit d-d transitions well below 10,000 cm1, assuming that the ligand field is not unusually strong. We have undertaken, therefore, a detailed investigation of the near-infrared absorption, CD, and magnetic circular dichroism (MCD) spectra of stellacyanin, plastocyanin, and azurin. Detailed analysis of these and analogous visible spectral data has allowed assignments to be made of both the d-d and charge transfer transitions. Interpretation of the charge transfer spectra has been aided further by the identification of the probable ligands of Cu(II) in bean plastocyanin. MATERIALS AND METHODSFrench bean plastocyanin (Phaseolus vulgaris) (18), azurin (Pseudomonas aeruginosa) (19), and stellacyanin (Rhus vernicifera) (20) were purified by standard methods. Near-infrared spectra were obtained on protein films to minimize interference by water absorption and permit a wide variation of temperature. The protein solutions were dialyzed against deionized distilled water and concentrated, first by pressurized membrane ultrafiltration and then, by placing drops on a plexiglas disk in a metal dessicator over Drierite. After three of four drops had been successively concentrated (ten to fifteen for the thick films), the film was prepared'by transferring the disk to a dessicator containing a saturated potassium acetate solution. This controlled humidity allowed the film to form slowly, thereby preventing most of the cracking caused by rapid removal of excess water. The drying process was halted by transfer of the film to a dessicator charged with a saturated sodium hydrogen phosphate solution.The near-infrared CD and MCD spectra were obtained with samples run in deuterated phosphate buffer in order to extend the spectral range to about 1850 nm. Although the infrared overtones of water do not give rise to a measureable CD spectrum, an absorbance of 1.0 or greater diminished the light level to the limit of detector response. The deuterated protein solutions were prepared by evaporating unbuffered, concentrated aqueous solutions in a Drierite dessicator as described above. The films were then transferred to a

Resonance Raman spectra of oxidized spinach ferredoxin and adrenodoxin show six and seven bands in the Fe-S stretching region (280-430 cm"1). Reconstitution of the proteins with labile 34S using rhodanese and 34SS032" produced isotope shifts allowing identification of all four Fe2S2 bridging modes and two (three for adrenodoxin) of the four expected Fe-S(Cys) terminal modes. Similar spectra are observed for the analogue complexes Fe2S2(S2-o-xyl)22~(S2-o-xyl = o-xylylenedithiolate) and Fe2S2Cl42", but they show the expected Dih selection rules, and IR-active modes are absent or weak in the Raman spectra.All eight (bridging plus terminal) stretching modes were located via IR as well as Raman spectroscopy and were calculated with reasonable accuracy by using a Urey-Bradley force field scaled to the crystallographically determined interatomic distances. The spinach ferredoxin and Fe2S2(S2-oxyl)22~frequencies are very similar, but adrenodoxin shows appreciably different terminal frequencies, suggestive of conformational differences of the ligated cysteines. The strong activation of IR modes in the protein Raman spectra implies a protein-induced inequivalence of the two ends of the Fe2S2 complex, which may contribute to the known localization of the added electron in the reduced form. This influence is suggested to be due to enhanced H bonding to the cysteine sulfur atoms at one end of the complex, consistent with available crystal structure data. Reduction of adrenodoxin shifts the bridging frequencies 16-24 cm'1 to lower frequency, consistent with the expected weakening of the bridging bonds. The terminal modes, however, were unobserved, plausibly due to a loss of enhancement associated with weakened terminal S -* Fe charge transfer intensity, except for a weak band at 308 cm"1. At higher frequencies, 550-850 cm'1, the protein spectra showed weak bands associated with the Fe-S overtone and combination levels. Previously reported spin-ladder Raman bands of adrenodoxin and spinach ferredoxin were not observed and are attributed to artifacts produced by laser-induced protein damage. A very broad (~200 cm'1) and weak feature at ~1000 cm"1 in the adrenodoxin spectrum might possibly be due to the S = 0 -1> 1 electronic transition of the spin-coupled Fe3+ ions.

enkephalin, eluted at 165 min, was collected and accounted for 92% of the starting material.(B) Bovine Growth Hormone Fragment (128)(129)(130)(131). The tetrapeptide Boc-Glu(OBzl)-Asp(OBzl)-Gly-Thr(Bzl)-OCH2-resin was synthesized from Boc-Thr(Bzl)-OCH2-resin (5 g, 0.22 mmol/g). Samples of 100-250 mg each were treated with different HF procedures as required.The results (Table VIII) were quantitated by ion-exchange chromatography on an AA-15 column (Beckman, 0.9 X 54 cm) attached on a Beckman 120B amino acid analyzer, eluted with pH 3.20 citrate buffer at 59 °C. The elution times of the peptides were /3-peptide 53 min, -peptide 70 min, and imide 135 min (Scheme VI).(C) Pentagastrin Amide. The pentapeptide Boc-Gly-Trp(For)-Met-(0)-Asp(0Bzl)-Phe-NH-CH(C6H5)-C6H4-0C0CH2-resin (3), was synthesized from a multidetachable benzhydrylamine-resin, prepared according to ref 46, 5 g, 0.18 mmol/g. Samples of 100-250 mg each were treated with different HF procedures as required. The results are summarized in Tables XII and XIII.

The nature of the dioxygen-iron bond in Hb02 and 02* is becoming clarified through studies of crystalline model compounds. Recently we reported the synthesis,2 magnetic properties, Mossbauer spectra, and X-ray crystallographic structure3 for a reversibly formed iron(II) dioxygen complex, 1, derived from a "picket fence porphyrin" and an axial imidazole (Figure 1). This complex has an end-on angular Fe02 bond with Fe-0-0 136 (4)°, Fe-0 1.75 (0.02) A, 0-0 1.25 (0.08) Á, and Fe-N (Á-Me-Im) 2.07 (0.02) A, in accord with Contribution No. 4881,

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