The formation of stable fatty acid substrate complexes in prostaglandin H₂ synthase-1

We have developed a protocol to purify apo-ovine (o) prostaglandin endoperoxide H₂ synthase-1 (PGHS-1) to homogeneity from ram seminal vesicles. The resulting apo enzyme can then be reconstituted with Co³⁺-protoporphyrin IX instead of Fe³⁺-protoporphyrin IX to produce a native-like, but functionally inert, enzyme suitable for the production of enzyme:fatty acid substrate complexes for biophysical characterization. Co³⁺-protoporphyrin IX reconstituted oPGHS-1 (Co³⁺-oPGHS-1) displays a Soret band at 426 nm that shifts to 406 nm upon reduction. This behavior is similar to that of cobalt-reconstituted horseradish peroxidase and myoglobin and suggests, along with resonance Raman spectroscopy, that the Co³⁺-protoporphyrin IX group is one in a six-coordinate, cobalt(III) state. However, Co³⁺-oPGHS-1 does not display cyclooxygenase or peroxidase activity, nor does the enzyme produce prostaglandin products when incubated with [1-¹⁴C] arachidonic acid. The cocrystallization of Co³⁺-oPGHS-1 and the substrate arachidonic acid (AA) has been achieved using sodium citrate as the precipitant in the presence of the nonionic detergent N-octyl-β-D-glucopyranoside. Crystals are hexagonal, belonging to the space group P6₅22, with cell dimensions of a = b = 181.69 Å and c = 103.74 Å, and a monomer in the asymmetric unit. GC-MS analysis of dissolved crystals indicates that unoxidized AA is bound within the crystals. (C) 2000 Academic Press.