Reactive intermediates and structure determination of heme proteins studied by using resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy is particularly useful for studies involving metalloporphyrin enzymes because of the enhancement effect from the porphyrin scattering due to its strong absorption. The RR technique has been used to study the reaction of cytochrome c oxidase (CcO) with O2, characterize the peroxidase site of prostanglandin H synthase-1 (PGHS-1), and the study of histidine rich protein II (HRichP-II) and the effects of the anti-malarial drug chloroquine (CQ) on the heme-bound structure.;There is a consensus on the vibrations of several of the intermediates that are formed in the reaction of the fully reduced CcO and O2. However, due its transient nature, there has been a controversy as to whether the first intermediate that is produced after the O--O bond cleavage, Pr, can be detected by time resolved RR. In this study, the P r intermediate has been detected with at about 50 mus after the O 2 binding. This species shows a vibration at 804 cm-1 and is assigned to a ferryl-oxo heme intermediate. In addition, careful examination of the spectra produced by 10-ns laser pulses revealed a new vibration at 590/543 cm-1 for the reaction between fully reduced CcO and 16O2/18O2. This mode parallels the formation of the 786 cm-1 band, from the ferryl-oxo unit of Compound F. The intensity of the 590 cm-1 mode increases at higher probe power at the expense of the 786 cm -1 mode, indicative of a photoreaction involving this ferryl-oxo compound.;A strong bond between the heme iron and the imidazolate at the proximal position is believed to play an essential role in the mechanism of O--O bond cleavage by most known peroxidases. However in PGHS-1, the proximal ligand plays a more important role in terms of the stability of the intermediates that are formed during the peroxidase reaction. Since, a tyrosyl radical is formed in PGHS-1, the weaker proximal histidine makes the ferryl-oxo intermediate in PGHS-1 (Compound I) a better oxidant than that of other peroxidases that are involved instead in the oxidation of substrates.;Malaria infects the red blood cells and digests hemoglobin and uses an enzyme, HRichP-II, to aggregate the free hemes into an insoluble polymer called hemozoin. This protein can bind up to 50 hemes to form a stable heme-bound protein. The Raman spectroscopic results indicate that the majority of the hemes, in the 50-heme-bound HRichP-II, are 6-coordinate, low-spin with two histidines as the axial ligands. The addition of the antimalarial agent, CQ, to heme-bound HRichP-II results in the formation a free heme-CQ complex. Therefore, CQ may act by sequestering the heme from the heme-bound HRichP-II to form a heme-CQ complex. (Abstract shortened by UMI.).