We report 441.6 nm excitation resonance Raman spectra of oxidized and reduced monomeric heme a-imidazole, cytochrome oxidase-exogenous ligand complexes in various redox states, and alkaline denatured oxidase. These data show that, in reduced oxidase, the cytochrome a3 Raman spectrum has bands at 215, 364, 1230, and 1670 cm-1 not observed in the cytochrome a spectrum. The appearance of these bands in the reduced cytochrome a3 spectrum is due to interactions between the heme a of cytochrome a3 and its protein environment and not to intrinsic properties of heme a. These interactions are pH sensitive and strongly influence the vibrational spectra of both heme a groups. We assign the 1670-cm-1 band to the heme a formyl substituent and propose that the intensity of the 1670 cm-1 is high for reduced cytochrome a3 because the C==O lies in the porphyrin plane and is very weak for oxidized and reduced cytochrome a, oxidized cytochrome a3, and oxidized and reduced heme a-imidazole because the C==O lies out of the plane. We suggest that movement of the C==O in and out of the plane explains the ligand induced spectral shift in the optical absorption spectrum of reduced cytochrome a3. Finally, we confirm the observation of Adar & Yonetani (private communication) that, under laser illumination, resting oxidase is photoreactive.

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The two-iron ferredoxins from spinach, parsley, Azotobacter vinelandii, Closlridium pasteurianum and the pig adrenal cortex were investigated by M6ssbauer spectroscopy at temperatures from 4 to 256°K and in magnetic fields up to 46 kGauss. Computational programs were devised to allow comparison of the experimental data with computer-simulated spectra in order to facilitate identification of the experimental spectral detail with specific M6ssbauer spectroscopic parameters (quadrupole splittings, isomer shifts and nuclear hyperfine and nuclear Zeeman interactions). The results of the analysis permit the following properties of the active center to be established directly as the result of these experiments: I. In the oxidized forms of the proteins, each iron is in the high spin (S 5/2) ferric state, spin-coupled to produce a resultant molecular diamagnetism for the protein at temperatures below Ioo°K. 2. In tile reduced state of the protein, the active center contains a single ferric site, retaining many properties of the ferric iron in the oxidized protein, but spin-Abbreviation: ENDOR, electron nuclear double resonance. * This is one of a series of papers describing the electronic properties of spinach ferredoxin and other iron-sulfur proteins; related publications contain the results of experiments on the electron nuclear double resonance (called i)9, magnetic susceptibility (called lIl) 1° and infrared, optical and circular dichroism (called IV) is spectra. An integrated interpretation of all our data in terms of a specific model for these proteins will be published separately. The work reported in this paper (called 11

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