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.