Songzhou Hu

Heme-protein interactions in cytochrome c peroxidase revealed by site-directed mutagenesis and resonance Raman spectra of isotopically labeled hemes

Isotope labeling has been used to assign the resonance Raman spectra of cytochrome c peroxidase, expressed in Escherichia coli [CCP (MKT)], and of the D235N site mutant. 54Fe labeling establishes the coexistence of two separate bands (233 and 246 cm-1), arising from the stretching of the bond between the Fe atom and the proximal histidine ligand, His175. These are assigned to tautomers of the H-bond between the His175 imidazole NΓH proton and the Asp235 carboxylate side chain: In one tautomer the proton resides on the imidazole while in the other the proton is transferred to the carboxylate. When Asp235 is replaced by Asn, the H-bond is lost, and the Fe-His stretching frequency is markedly lowered. Two new RR bands are produced, at 205 and 185 cm-1, as a result of coupling between the shifted Fe-His vibration and a nearby porphyrin mode; the two bands share the 54Fe sensitivity expected for Fe-His stretching. C=C stretching and CβC=C bending vibrations have been separately assigned to the 2- and 4-vinyl groups of the protoheme prosthetic group via selective vinyl deuteration. In the acid form of the enzyme, the frequencies coincide for the two vinyl groups, at 1618 cm-1 for the C=C stretch, and at 406 cm-1 for the CβC=C bend. However, the 2-vinyl frequencies are elevated in the alkaline form of the enzyme, to 1628 cm-1 for C=C stretching, and to 418 cm-1 for CβC=C bending, while the 4-vinyl frequencies remain unshifted. Thus, the acid-alkaline transition involves a protein conformation change that specifically perturbs the 2-vinyl substituent. This perturbation might be a reorientation of the vinyl group, or an alteration of the porphyrin geometry that affects the porphyrin-vinyl coupling. The perturbation is attenuated when CO is bound to the enzyme; the C=C frequency is then unaffected in the alkaline form, while the CβC=C bending frequency is shifted to a smaller extent (412 cm-1). This attenuation is probably linked to inhibition of distal histidine binding to the heme Fe in the alkaline form when the CO is bound.

Variable forms of soluble guanylyl cyclase: protein-ligand interactions and the issue of activation by carbon monoxide.

1, the resting Fe(II) state is mainly 6-coordinate and low-spin, and the CO adduct has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment. In contrast, the protein sGC2 is 5-coordinate, high-spin in the resting state, and the CO adduct has perturbed vibrational frequencies indicative of a negatively polarizing residue in the binding pocket. The differences may result from the need to reconstitute sGC1 or different isolation procedures for sGC1 versus sGC2. However, both sGC1 and sGC2 are activated by the same mechanism, namely displacement of the proximal histidine ligand upon NO binding, and neither one is activated by CO. If CO is an activator in vivo, some additional molecular component is required.

MODELING THE BONDING CHANGES IN CHLOROPHYLL CATION RADICALS : RESONANCE RAMAN SPECTROSCOPY OF NICKEL(II) METHYL PYROPHEOPHORBIDE A

Abstract Electrochemistry, UV-Vis absorption and resonance Raman (RR) spectra are reported for nickel methyl pyropheophorbide a (NiMPPh) and its cation radical, in order to help understand the structural changes upon oxidation in chlorophyll cation radicals. RR band shifts, and also the oxidation potential, are consistent with the highest occupied molecular orbital (HOMO) of NiMPPh being an A1u-like orbital, which interacts with the keto-carbonyl group on the isocyclic ring. Of particular interest is the upshift of the keto-carbonyl stretching mode in the NiMPPh cation radical. The strengthening of the carbonyl bond upon oxidation is consistent with the expected anti-bonding character of the A1u-like orbital with respect to the keto CO bond.

Singlet and Triplet (π, π*) Excited States of Zinc(II) Octaalkylporphyrins: A Time-Resolved Resonance-Raman Study

The excited states of porphyrins and hydroporphyrins act as primary intermediates in processes ranging from biological energy transfer and electron transfer to photocatalysis and photodynamic therapy. We report resonance Raman (RR) spectra of the singlet S1 and triplet T1 excited states of zinc(II) octaethylporphyrin (ZnOEP) and zinc(II) etioporphyrin-I (ZnEtio), using picosecond and nanosecond RR techniques. Picosecond time-resolved resonance Raman experiments were performed using 532 nm photoexcitation pulses and 436 nm probe pulses (both 100 ps, 50 Hz); the experimental apparatus consisted of a mode-locked Nd:YAG laser (Coherent) in combination with a Nd:YAG regenerative amplifier (Continuum) and an H2 cell for stimulated Raman shifting. The apparatus used for nanosecond time-resolved RR experiments has been described previously [1]. Porphyrin samples were prepared in a nitrogen atmosphere at a concentration of 0.5 mM (tetrahydrofuran solvent).