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To evaluate the importance of the axial ligand in blue Cu centers, the electronic structure of a three-coordinate model compound LCuSCPh3 (2, L = β-diketiminate ligand) is defined using low-temperature absorption, magnetic circular dichroism (MCD), X-ray absorption spectroscopy (XAS), and resonance Raman (rR) profiles coupled with density functional calculations. Using these excited-state spectroscopic methods the electronic structure of 2 is compared to that of a four-coordinate blue Cu model compound LCuSCPh3 (1, L = tris(pyrazolyl)hydroborate ligand) and the three-coordinate blue Cu center in fungal laccase. The spectral features of 2 are substantially altered from those of 1 and reflect a trans influence of the β-diketiminate ligand that involves a strong interaction with the Cu dx2-y2 orbital, concomitant with a decreased S pπ interaction with the Cu dx2-y2 orbital. The lack of an axial ligand coupled with the influence of this equatorial donor leads to many of the perturbed spectral features of 2 re...

A tris(pyrazolyl)hydroborate triphenylmethylthiolate Cu(II) model complex (1) that reproduces structural and spectroscopic features of active sites of blue Cu proteins is characterized using low-temperature absorption, magnetic circular dichroism (MCD), X-ray absorption (XAS), and resonance Raman (rR) spectroscopies combined with DFT calculations to define its electronic structure. The electronic structure of 1 is further related to the oxidized Cu site in plastocyanin. The key spectral differences relative to plastocyanin include an increase in the intensity of the S pπ → Cu CT band and a decrease in the absorption intensity at ∼450 nm. The energies of d → d transitions in 1 decrease relative to plastocyanin, which reflects the more tetrahedral geometry of 1. S K-edge XAS measurements demonstrate a more covalent thiolate interaction in the HOMO of 1 (52% S p) than in plastocyanin (38% S p). The effects of the high thiolate covalency on the absorption and Raman spectral features for 1 are evaluated. Addit...

We have performed continuous-wave electron paramagnetic resonance (CW-EPR) and electron spin echo electron nuclear double resonance (ESE-ENDOR) experiments on the multiline form of the S2-state of untreated, MeOH-treated, and ammonia-treated spinach photosystem II (PS II) centers. Through simultaneously constrained simulations of the CW-EPR and ESE-ENDOR data, we conclude that four effective 55Mn hyperfine tensors (AX, AY, AZ) are required to properly simulate the experimental data [untreated and MeOH-treated PS II centers (MHz): −232, −232, −270; 200, 200, 250; −311, −311, −270; 180, 180, 240; ammonia-treated PS II centers (MHz): 208, 208, 158; −150, −150, −112; 222, 222, 172; −295, −315, −390]. We further show that these effective hyperfine tensors are best supported by a trimer/monomer arrangement of three Mn(IV) ions and one Mn(III) ion. In this topology, MnA, MnB, and MnC form a strongly exchange coupled core (JAB and JBC < −100 cm-1) while MnD is weakly exchange coupled (JCD) to one end of the trin...

A variety of spectroscopic techniques combined with density functional calculations are used to describe the electronic structure of the nonaxially ligated, trigonal planar type 1 copper site in three fungal laccases with substantially different type 1 copper reduction potentials. These methods are also applied to a mutant of the high-potential Polyporus pinsitis laccase in which the nonligating axial phenylalanine (Phe) is changed to methionine (Met). Optical absorption, circular dichroism, and magnetic circular dichroism spectroscopies of all three fungal laccases reveal that, relative to the classic blue copper protein plastocyanin, the ligand field strength at the type 1 Cu center and the oscillator strength of the charge-transfer transitions increase. Resonance Raman spectra show that the envelope of Cu−S(Cys) stretching bands is shifted to higher energy in the fungal laccases, implying a stronger Cu−S(Cys) bond. Differences in the EPR spectra of the fungal laccases and plastocyanin are found to resu...

The electronic structures of the perturbed blue copper proteins stellacyanin (STC) and cucumber basic protein (CBP, also called plantacyanin, PNC) are defined relative to that of the well-understood “classic” site found in plastocyanin (PLC) by combining the results of low-temperature optical absorption, circular dichroism, and magnetic circular dichroism spectra with density functional calculations. Additionally, absorption and magnetic circular dichroism spectra of Alcaligenes denitrificans wild-type and M121Q azurin are presented and compared to PLC and STC, respectively. These studies show that the principal electronic structure changes in CBP/PNC, with respect to PLC, are a small shift of the ligand field transitions to higher energy and a rotation of the Cu dx2-y2 half-filled HOMO which increases the pseudo-σ and decreases the π interactions of the cysteine (Cys) sulfur with Cu dx2-y2 and, in addition, mixes some methionine (Met) sulfur character into the HOMO. The geometrical distortion responsible...

X-ray absorption (XAS), UV−visible, electron paramagnetic resonance (EPR), and electron spin echo envelope modulation (ESEEM) spectroscopies have been used to characterize the interaction of azide and cyanide with the Mn(III)(μ-O)2Mn(IV) site in superoxidized Mn catalase. The addition of azide causes no significant change in the X-ray absorption near edge structure (XANES) region and only minor changes in the extended X-ray absorption fine structure (EXAFS) spectra, consistent with only minimal changes in Mn−ligand geometry. In contrast, addition of either azide or cyanide causes an approximately 3-fold increase in the intensity of the visible absorption bands and a small (approximately 4%) decrease in the Mn hyperfine coupling. Anion-binding titrations indicate cooperativity in anion binding. ESEEM experiments on the azide- and cyanide-free enzyme reveal hyperfine (A = 2.88 MHz) and electric quadrupolar couplings (e2qQ = 2.29 MHz and η = 0.58) for a single 14N nucleus of the protein. These parameters are...

Interactions of water and methanol with a mixed valence Mn(III)Mn(IV) complex are explored with 1H electron spin echo (ESE)-electron nuclear double resonance (ENDOR) and 1H and 2H ESE envelope modulation (ESEEM). Derivatives of the (2-OH-3,5-Cl2-SALPN)2 Mn(III)Mn(IV) complex are ideal for structural and spectroscopic modeling of water binding to multinuclear Mn complexes in metalloproteins, specifically photosystem II (PSII) and manganese catalase (MnCat). Using ESE-ENDOR and ESEEM techniques, 1H hyperfine parameters are determined for both water and methanol ligated to the Mn(III) ion of the complex. The protons of water directly bound to Mn(III) are inequivalent and exhibit roughly axial dipolar hyperfine interactions (Tdip = 8.4 MHz and Tdip = 7.4 MHz), permitting orientations and radial distances to be determined using a model where the proton experiences a point dipole interaction with each Mn ion. General equations are given for the components of the rhombic dipolar hyperfine interaction between a p...

Analysis of {sup 55}Mn electron spin echo-electron nuclear double resonance (ESE-ENDOR) spectra obtained on a dinuclear mixed valence Mn(III)Mn(IV) complex [di-{mu}-oxotetrakis(2, 2`-bipyridine)dimanganese(III,IV)] (1) reveals the hyperfine and nuclear quadrupolar parameters for the spin I=5/2 {sup 55}Mn nucleus of both Mn(III) and Mn(IV) ions. The {sup 55}Mn ESE-ENDOR data obtained on the g = 2 Mn multiline EPR signal of the S{sub 2} state of the photosystem II oxygen-evolving complex demonstrate that this EPR signal cannot arise from a dinuclear Mn(III)-Mn(IV) center. The ENDOR spectra are consistent with a tetranuclear Mn cluster origin for the photosystem II multiline EPR signal. 75 refs., 7 figs., 2 tabs.

The photochemical lability of peptides is poorly understood, largely because of the lack of product data. In the present study, product analyses have been carried out following the photolyses in aqueous solution of selected glycyl dipeptides (Gly-Gly, DL-AIa-GIy, L-VaI-GIy, L-Pro-Gly, L-Phe-Gly, and Gly-L-Phe), L-prolyl-L-phenylalanine, and L-phenylalanyl-L-proline. Efficient deamination and decarboxylation of aliphatic dipeptides generate thermal precursors of simple amides in a photoinduced electron-transfer process involving the peptide bond. An analogous pathway in the photodegradation of phenylalanyl peptides suffers competition from other types of reaction.

Detailed electronic and geometric structural descriptions of the blue copper sites in wild-type (WT) stellacyanin and its Q99M and Q99L axial mutants have been obtained using a combination of XAS, resonance Raman, MCD, EPR, and DFT calculations. The results show that the origin of the short Cu-S(Cys) bond in blue copper proteins is the weakened axial interaction, which leads to a shorter (based on EXAFS results) and more covalent (based on S K-edge XAS) Cu-S bond. XAS pre-edge energies show that the effective nuclear charge on the copper increases going from O(Gln) to S(Met) to no axial (Leu) ligand, indicating that the weakened axial ligand is not fully compensated for by the increased donation from the thiolate. This is further supported by EPR results. MCD data show that the decreased axial interaction leads to an increase in the equatorial ligand field, indicating that the site acquires a more trigonally distorted tetrahedral structure. These geometric and electronic structural changes, which result from weakening the bonding interaction of the axial ligand, allow the site to maintain efficient electron transfer (high H(DA) and low reorganization energy), while modulating the redox potential of the site to the biologically relevant range. These spectroscopic studies are complemented by DFT calculations to obtain insight into the factors that allow stellacyanin to maintain a trigonally distorted tetrahedral structure with a relatively strong axial Cu(II)-oxygen bond.