Angel J. Di Bilio

Tryptophan-Accelerated Electron Flow Through Proteins

Energy flow in biological structures often requires submillisecond charge transport over long molecular distances. Kinetics modeling suggests that charge-transfer rates can be greatly enhanced by multistep electron tunneling in which redox-active amino acid side chains act as intermediate donors or acceptors. We report transient optical and infrared spectroscopic experiments that quantify the extent to which an intervening tryptophan residue can facilitate electron transfer between distant metal redox centers in a mutant Pseudomonas aeruginosa azurin. CuI oxidation by a photoexcited ReI-diimine at position 124 on a histidine(124)-glycine(123)-tryptophan(122)-methionine(121) β strand occurs in a few nanoseconds, fully two orders of magnitude faster than documented for single-step electron tunneling at a 19 angstrom donor-acceptor distance.

Tricarbonyl(1,10-phenanthroline) (imidazole) rhenium(I): a powerful photooxidant for investigations of electron tunneling in proteins☆

Abstract The structure of [Re(CO)3(phen)(im)]2SO4·4H2O has been determined by X-ray crystallography. The yellow crystals are orthorhombic, space group Pccn (No. 56), with a=17.456(6), b=18.194(5), c=12.646(4) A , R=0.063 for F o 2 >0, R=0.032 for F o 2 >3σ . The compound, which also has been characterized by IR, 1H NMR, and UVVis spectroscopies, exhibits room temperature luminescence in aqueous solution (τ=120 ns) as well as reversible oxidation and reduction in acetonitrile solution (1.85 and −1.30 V versus SCE). The redox properties of the excited state of the complex (E 0 ( Re +∗/0 = 1.2; E 0 ( Re 2+/+∗ ) = −0.7 V ) are being exploited in studies of laser-induced electron tunneling in Re(CO)3(phen)(histidine)-modified proteins.

A Ferrous-Triapine complex mediates formation of reactive oxygen species that inactivate human ribonucleotide reductase.

Ribonucleotide reductase plays a central role in cell proliferation by supplying deoxyribonucleotide precursors for DNA synthesis and repair. The holoenzyme is a protein tetramer that features two large (hRRM1) and two small (hRRM2 or p53R2) subunits. The small subunit contains a di-iron cluster/tyrosyl radical cofactor that is essential for enzyme activity. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a new, potent ribonucleotide reductase inhibitor currently in phase II clinical trials for cancer chemotherapy. Ferric chloride readily reacts with Triapine to form an Fe(III)-(3-AP) complex, which is reduced to Fe(II)-(3-AP) by DTT. Spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide prove that Fe(II)-(3-AP) reduces O2 to give oxygen reactive species (ROS). In vitro activity assays show that Fe(II)-(3-AP) is a much more potent inhibitor of hRRM2/hRRM1 and p53R2/hRRM1 than Triapine. Electron paramagnetic resonance measurements on frozen solutions of hRRM2 and p53R2 show that their tyrosyl radicals are completely quenched by incubation with Fe(II)-(3-AP). However, the enzyme activity is maintained in protein samples supplemented with catalase alone or in combination with superoxide dismutase. Furthermore, catalase alone or in combination with superoxide dismutase markedly decreases the antiproliferative effect of Triapine in cytotoxicity assays. These results indicate that Triapine-induced inhibition of ribonucleotide reductase is caused by ROS. We suggest that ROS may ultimately be responsible for the pharmacologic effects of Triapine in vivo. [Mol Cancer Ther 2006;5(3):586–92]

Electron transfer in ruthenium-modified proteins

Photochemical techniques have been used to measure the kinetics of intramolecular electron transfer in Ru(bpy)2(im)(His)2+-modified (bpy = 2,2′-bipyridine; im = imidazole) cytochromec and azurin. A driving-force study with the His33 derivatives of cytochromec indicates that the reorganization energy (γ) for Fe2+→Ru3+ ET reactions is 0.8 eV. Reductions of the ferriheme by either an excited complex,*Ru2+, or a reduced complex, Ru+, are anomalously fast and may involve formation of an electronically excited ferroheme. The distance dependence of Fe2+→Ru3+ and Cu+→Ru3+ electron transfer in 12 different Ru-modified cytochromes and azurins has been analyzed using a tunneling-pathway model. The ET rates in 10 of the 12 systems exhibit an exponential dependence on metal-metal separation (decay constant of 1.06 å−1) that is consistent with predictions of the pathway model.

Electron tunneling in biological molecules

Electron transfers in photosynthesis and respiration commonly occur between protein-bound prosthetic groups that are separated by large molecular distances (often greater than 10A). Although the electron donors and acceptors are expected to be weakly coupled, the reactions are remarkably fast and proceed with high specificity. Tunneling timetables based on analyses of Fe^(2+)/Cu^+ to Ru^(3+) electron-transfer rates for Ru-modified heme and copper proteins reveal that the structure of the intervening polypeptide can control these distant donor-acceptor couplings. Multistep tunneling can account for the relatively rapid Cu^+ to Re^(2+) electron transfer observed in Re-modified azurin.

Deeply Inverted Electron-Hole Recombination in a Luminescent Antibody-Stilbene Complex

The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedented ability to produce bright luminescence and has been used as a biosensor in various applications. We show that the prolonged luminescence is not stilbene fluorescence. Instead, the emissive species is a charge-transfer excited complex of an anionic stilbene and a cationic, parallel π-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody.

Relaxation dynamics of Pseudomonas aeruginosa Re(I)(CO)3(alpha-diimine)(HisX)+ (X = 83, 107, 109, 124, 126)Cu(II) azurins.

Photoinduced relaxation processes of five structurally characterized Pseudomonas aeruginosa Re(I)(CO)(3)(alpha-diimine)(HisX) (X = 83, 107, 109, 124, 126)Cu(II) azurins have been investigated by time-resolved (ps-ns) IR spectroscopy and emission spectroscopy. Crystal structures reveal the presence of Re-azurin dimers and trimers that in two cases (X = 107, 124) involve van der Waals interactions between interdigitated diimine aromatic rings. Time-dependent emission anisotropy measurements confirm that the proteins aggregate in mM solutions (D(2)O, KP(i) buffer, pD = 7.1). Excited-state DFT calculations show that extensive charge redistribution in the Re(I)(CO)(3) --> diimine (3)MLCT state occurs: excitation of this (3)MLCT state triggers several relaxation processes in Re-azurins whose kinetics strongly depend on the location of the metallolabel on the protein surface. Relaxation is manifested by dynamic blue shifts of excited-state nu(CO) IR bands that occur with triexponential kinetics: intramolecular vibrational redistribution together with vibrational and solvent relaxation give rise to subps, approximately 2, and 8-20 ps components, while the approximately 10(2) ps kinetics are attributed to displacement (reorientation) of the Re(I)(CO)(3)(phen)(im) unit relative to the peptide chain, which optimizes Coulombic interactions of the Re(I) excited-state electron density with solvated peptide groups. Evidence also suggests that additional segmental movements of Re-bearing beta-strands occur without perturbing the reaction field or interactions with the peptide. Our work demonstrates that time-resolved IR spectroscopy and emission anisotropy of Re(I) carbonyl-diimine complexes are powerful probes of molecular dynamics at or around the surfaces of proteins and protein-protein interfacial regions.

Electronic absorption spectra of M(II)(Met121X) azurins (MCo, Ni, Cu; XLeu, Gly, Asp, Glu): charge-transfer energies and reduction potentials

Abstract Electronic absorption spectra of the Co(II) and Ni(II) derivatives of Met121X (XLeu, Gly, Asp, Glu) azurin mutants have been measured. Coordination of carboxylate to the metal ion is indicated by LF and LMCT band shifts in the Met121Glu proteins. The relatively low reduction potentials of the Cu(II)(Met121X) (XAsp, Glu) azurins accord with the LMCT energies of the corresponding Co(II) derivatives.

X-Ray crystal structures and electron spin resonance spectroscopic characterization of mixed-ligand chromium(III) complexes with L-aspartate or pyridine-2,6-dicarboxylate and 1,10-phenanthroline or 2,2′:6′,2″-terpyridyl

The crystal and molecular structure of two newly prepared chromium(III) complexes [Cr(L-asp)(phen)(H2O)]NO3·2H2O 1[L-asp =L-aspartate(2–), phen = 1,10-phenanthroline] and [Cr(terpy)(pydca)][Cr(pydca)2]·4H2O 3(pydca = pyridine-2,6-dicarboxylate, terpy = 2,2′:6′,2′′-terpyridyl) were determined by means of X-ray diffraction. Crystals of 1 are triclinic, space group P, with a= 11.793(5), b= 10.507(5), c= 9.258(5)A, α= 111.43(3), β= 86.44(3) and γ= 111.71(3)°. Crystals of 3 are triclinic, space group P, with a= 19.100(5), b= 12.874(5), c= 7.575(5)A, α= 86.16(3), β= 95.10(3) and γ= 96.62(3)°. The co-ordination around the Cr atom in both 1 and 3 is distorted octahedral, the sixth position of 1 being occupied by a water molecule. Crystals of 3 are built up of two different ionic units. ESR spectra were run on magnetically dilute powders and frozen solutions, and the values of the spin-Hamiltonian parameters obtained by computer simulation. The similarity between these parameters for the chromium(III) species in powders and glasses suggests that the solution species possess the same kind of distortion found for the solid complexes.

EPR study on vanadyl and vanadate ion retention by a thermotolerant yeast

Abstract The retention of vanadyl(IV) and vanadate(V) ions by Hansenula polymorpha , a thermotolerant yeast, has been studied by EPR spectroscopy. Vanadyl ions were retained as mobile complexes associated with relatively low molecular weight ligands as well as immobilized complexes formed with high molecular weight residues, the donor atoms of which are probably oxygens or deprotonated hydroxyls. The distribution of vanadyl ions between mobile and immobilized complex species is a function of the metal concentration as well as of the contact time. Vanadate ions were reduced by cellular components to vanadyl ions.