The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 °C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1= 1.4 × 107 M−1 s−1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+· FeIVO). Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2= 65 s−1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome‐c peroxidase, was assigned to a two‐electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+· which was formed by an intramolecular electron transfer from tyrosine to the porphyrin‐π‐cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.

Purified prostaglandin H synthase (EC 1.14.994, reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high-spin ferric heme. Arachidonic acid with 02, as well as prostaglandin G2 or H 2 0 2 , decreased the spectrum of the native enzyme and concomitantly a doublet signal at g = 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half-life of less than 20 s at -12°C. From the conditions for the formation and the effect of inhibitors,

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Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a ubiquitin chain to a chaperone-presented client protein and thereby stimulates its proteasomal degradation. To gain further insight into the function of CHIP we isolated CHIP-containing protein complexes from human HeLa cells and analyzed their composition by peptide mass fingerprinting. We identified the Hsc70 cochaperone BAG-2 as a main component of CHIP complexes. BAG-2 inhibits the ubiquitin ligase activity of CHIP by abrogating the CHIP/E2 cooperation and stimulates the chaperone-assisted maturation of CFTR. The activity of BAG-2 resembles that of the previously characterized Hsc70 cochaperone and CHIP inhibitor HspBP1. The presented data therefore establish multiple mechanisms to control the destructive activity of the CHIP ubiquitin ligase in human cells.