The geometric and electronic structures of high-spin ferrous complexes of bleomycin ((FeBLM)-B-II) and a series of systematically perturbed BLM derivatives have been investigated by optical absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopies. The active site of the unmodified drug complex is six-coordinate with the coordination sphere completed by at least five endogenous ligands including the pyrimidine, imidazole, deprotonated amide, and secondary and primary amine functionalities with either the 3-O-carbamoyl substituent of the mannose sugar or solvent bound at the sixth site. This weak sixth ligand is the exchangeable site of exogenous small molecule binding. Perturbing the carbamoyl substituent alters the coordination environment of the metal and decreases the azide binding affinities of the perturbed complexes. This is correlated with altered DNA cleaving capabilities. Additionally, altering the binding of the axial primary amine significantly affects the iron coordination sphere as evidenced by reduced pi-back-bonding interactions specifically with the pyrimidine ligand. This pyrimidine pi-back-bonding appears to play a key role in mediating the electron density localized on the ferrous center, which contributes to the unique oxygen chemistry and reactivity exhibited by (FeBLM)-B-II relative to other non-heme iron sites. Oxygen binding to derivatives in which the beta-aminoalanine fragment has been removed leads to a high-spin ferric complex and no observed DNA strand scission, in contrast to the long-lived low-spin activated BLM intermediate that precedes DNA degradation.

Mononuclear non-heme iron active sites are present in a variety of enzymes involed in a wide range of important biological functions requiring dioxygen. These include superoxide dismutases, oxidases, extra- and intradiol dioxygenases, cis-dihydroxylases, pterin- and α-ketoglutarate-dependent hydroxylases, lipoxygenases, and bleomycin. Both the ferrous and ferric oxidation states are involved in catalysis for different enzymes in this class and substrate-and oxygen-bound intermediates have been observed. Much less is known about the active sites in these enzymes relative to the heme systems as the non-heme iron centers are less spectroscopically accessible, particularly at the ferrous oxidation level. The application of magnetic circular dichroism (MCD) spectroscopy has greatly advanced the level of understanding in these systems as this technique allows the direct observation of the ferrous d→d ligand field transitions, which are generally obscured by solvent and protein vibrations in optical absorption spectroscopy owing to the weak extinction coefficients and are often electron paramagnetic resonance silent due to relatively large ground-state sublevel splittings and fast relaxation times. The energies of the d→d transitions give the splitting of the excited-state eg orbitals which can be correlated with the coordination number and geometry at the ferrous center. In addition, the ground states of high-spin ferrous complexes are described as an s=2 spin manifold which undergoes axial zero-field splitting into Ms=±2, ±1, 0 components separated by 3D and D, respectively. The non-Kramers Ms=±2 doublet is lowest in energy for D<0, and is further rhombically split by an amount σ in the absence of a magnetic field. The unusual variable-temperature variable-field MCD saturation behavior observed for such systems can be interpreted by including the effects of σ as well as z-polarization, linear B-terms, and the population of low-lying excited states. The non-degenerate Ms=0 state is lowest in energy for D>0 and the resulting MCD saturation behavior can be analyzed by including the effects of off-axis Zeeman terms and z-polarized eletronic transitions. The spin Hamiltonian parameters obtained through analysis of the variable-temperature variable-field MCD saturation data are further interpreted in terms of the ligand field splitting of the ground-state t2g set of d-orbitals. This MCD methodology has been applied to several biologically relevant mononuclear non-heme ferrous systems to directly probe the active site geometric and electronic structures and to gain mechanistic information about their catalytic cycles. MCD spectroscopy has been used to study the native ferrous active site of phthalate dioxygenase and its interaction with substrate and exogenous ligands, which has previously been difficult to study due to the additional presence of a [2Fe-2S] Rieske cluster. The native form of soybean lipoxygenase exists as a mixture of species in solution that has been defined through the application of CD and MCD spectroscopies. This MCD methodology has also been used to elucidate the nature of the ferrous active site in bleomycin, which represents an important deviation from mononuclear non-heme iron enzymes in that it exhibits low-energy charge transfer transitions and performs chemistry similar to heme systems. The MCD methodology presented in this review has been employed to obtain molecular level insight into the catalytic mechanisms of these important enzyme systems and to understand the differences in active site geometric and electronic structures which relate to differences in oxygen reactivity.

The geometric and electronic structure of high-spin ferrous complexes of bleomycin (Fe(II)BLM) and the structural analog PMAH ([Fe(II)PMA](+), where PMAH is a macrocyclic ligand with pyrimidine, imidazole, deprotonated amide, and secondary and primary amine functionalities) have been investigated by optical (Abs) and X-ray (XAS) absorption, magnetic circular dichroism (MCD), and resonance Raman (rR) spectroscopies. From the excited state ligand field transition energies in the low-temperature MCD spectra, the XAS pre-edge shapes and intensities, and EXAFS analysis, solid [Fe(II)PMA](+) has been determined to have a five-coordinate, square-pyramidal geometry (E(dx2-y2) - E(dz2) = 6100 cm(-1)) with a short Fe-N bond (1.93 Angstrom), while in solution [Fe(II)PMA](+) binds a solvent molecule at the sixth position to form a distorted octahedral complex (E(dx2-y2) - E(dz2) = 2110 cm(-1)) with an expanded coordination sphere which still maintains one short Fe-N bond (2.00 Angstrom). Similar spectral features consistent with a six-coordinate geometry (E(dx2-y2) - E(dz2) = 2650 cm(-1)) are also present for Fe(II)BLM in solution, suggesting parallel ligation to the Fe2+ center in [Fe(II)PMA](+) including one relatively short Fe-N bond (2.06-2.08 Angstrom). The magnetic field and temperature dependence of the MCD intensity reveals an unusually small zero-field-splitting of the S = 2, M(s) = +/- 2 non-Kramers doublet ground state of Fe(II)BLM and [Fe(II)PMA](+) in solution (delta = 2.4 cm(-1)) indicating a large splitting of the d pi orbitals (E(dxz,yz) - E(dxy) = 800 and 950 cm(-1), respectively) resulting from strong metal-ligand pi-bonding interaction. The presence of moderately-intense, low-energy metal-to-ligand charge transfer (MLCT) transitions in the low-temperature Abs and MCD spectra of [Fe(II)PMA](+) and Fe(II)BLM represents an important deviation from other non-heme iron centers and reflects high covalency. The MLCT transition energies and intensities determine the degree of metal-ligand pi-backbonding which decreases along the series solid [Fe(II)PMA](+) > solution [Fe(II)PMA](+) > Fe(II)BLM. Assignment of these bands as iron(II) --> pyrimidine MLCT transitions is derived from the strong resonance enhancement of the pyrimidine normal modes at 680, 744, 1519, and 1542 cm(-1) in the [Fe(II)PMA](+) Raman spectrum, thereby implicating pyrimidine as the specific ligand associated with the large d pi splitting and short Fe-N bond. This pyrimidine pi-backbonding mediates the electron density localized on the Fe2+ center which contributes to the unique chemistry of Fe(II)BLM relative to other non-heme iron sites. This includes its ability to bind pi-acceptor exogenous ligands resulting in the conversion to a low-spin state and its formation of a long-lived oxygen intermediate.