In an effort to resolve an existing controversy involving the assignment of the bending fundamental [δ(FeCO)] of CO−ligated heme proteins, resonance Raman studies of the CO adducts of native hemoglobin (Hb), together with Hb containing selectively deuterated hemes, have been conducted. Hemes were utilized which were deuterated at the peripheral methyl positions (d12) or at the methine carbon positions (d4). Several CO isotope sensitive modes were observed in the low-frequency region (300−400 cm-1) by generating difference spectra from the absolute spectra of the natural abundance (NA) and doubly labeled 13C18O (DI) CO-Hb. These features included one at ∼370 cm-1. Deconvolution of the low-frequency region revealed that a 1−2 cm-1 shift of a heme mode at 367 cm-1 is responsible for this difference feature. In the mid-frequency spectral region (650−1300 cm-1), features which were previously suggested to be ascribable to combination bands and overtones involving the proposed ∼370 cm-1 δ(FeCO) fundamental showed sensitivity to heme deuteration, a fact which suggests that these modes are combinations involving heme modes rather than two internal fundamentals of the FeCO fragment. These results imply, therefore, that the observation of weak isotope-sensitive features in the 700−900 cm-1 region does not support the assignment of the ∼370 cm-1 feature to the δ(FeCO) fundamental, but these features are more reasonably interpreted to arise from combinations involving the relatively strong ν(Fe−CO) and lower frequency heme modes, an interpretation that is consistent with the long-standing assignment of the weak band near 580 cm-1 to the δ(FeCO) fundamental.

Low frequency resonance Raman (RR) spectra are reported for deoxy hemoglobin (Hb), its isolated subunits, its analogue bearing methine-deuterated hemes in all four subunits (Hb-d 4 ), and the hybrids bearing the deuterated heme in only one type of subunit, which are [␣ d4 ␤ h4 ] 2 and [␣ h4 ␤ d4 ] 2 . Analyzed collectively, the spectra reveal subunit-specific modes that conclusively document subtle differences in structure for the heme prosthetic groups in the two types of subunits within the intact tetramer. Not surprisingly, the most significant spectral differences are observed in the ␥ 7 mode that has a major contribution from out of plane bending of the methine carbons, a distortion that is believed to relieve strain in the high-spin heme prosthetic groups. The results provide convincing evidence for the utility of selectively labeled hemoglobin hybrids in unraveling the separate subunit contributions to the RR spectra of Hb and its various derivatives and for thereby detecting slight structural differences in the subunits.

Bleomycin is an antitumor agent whose cytotoxicity is dependent on its ability to bind DNA in the nucleus and effect double-stranded DNA cleavage, which is difficult for the cell to repair. In order for this DNA cleavage to occur, bleomycin must, through a series of reactions, form a low-spin Fe(III) complex, the putative "activated" form of the drug, HOO-Fe(III)bleomycin. The relative strengths of the bonds in the Fe(III)-OOH linkage have not been determined due to the weakness of the hydroperoxo-to-iron(III) charge-transfer transition. The much more stable HOO-Co(III)bleomycin has often been studied as a structural analogue of HOO-Fe(III)bleomycin, and hence, an understanding of the relative bond strengths in the Co-OOH linkage may serve to enhance our understanding of the analogous Fe-OOH linkage. In this report, we present resonance Raman data that identify two important vibrational modes in the Co-OOH linkage, the stretching modes, nu(Co-OOH) and nu(O-OH). Both of these vibrational modes were found to be unperturbed by complexation of the drug with calf thymus DNA. Advantage was also taken of the isostructural realtionship between Fe-bleomycin and Co-bleomycin to analyze and assign the high-frequency modes for HOO-Co(III)bleomycin and Co(III)bleomycin (A(2) and B(2)). These data could be useful for future studies of photoactivated Co-bleomycin and Co-bleomycin analogues in an attempt to characterize oxygen-independent DNA damage pathways.

The resonance Raman spectra of the adducts of cytochrome c peroxidase with four cyanide isotopomers (l2Cl4N-, tially linear conformer whose v(Fe-C) and S(FeCN) modes occur at 445 cm-and 407 cm-', respectively (at pH 10) and a bent conformer which exhibits two modes at 355 cm-' and 445 cm-I (at pH 10). At pH values below 7, the linear form v(Fe-C) shifts to -455 Ern-' in response to protonation of the distal histidyl imidazole (which strengthens the Fe-C bond). The stretching mode of the bent form experiences only a slight shift. This smaller shift of the bent form is consistent with the proposal that the bent form originates as a consequence of hydrogen bond formation with the off-axis proton donor group of arginine (which is protonated at both pH values). The overall spectral response to pH changes reflects interactions with the bound ligand which may be important for heterolytic bond cleavage. 1 3~1 4~-12CiSN-and 13 1s 9 C N -) are presented. The spectra reveal the presence of two conformers, an essen-