Peroxidase activity in prostaglandin endoperoxide H synthase-1 occurs with a neutral histidine proximal heme ligand.
Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) convert arachidonic acid to prostaglandin H(2) (PGH(2)), the committed step in prostaglandin and thromboxane formation. Interaction of peroxides with the heme sites in PGHSs generates a tyrosyl radical that catalyzes subsequent cyclooxygenase chemistry. To study the peroxidase reaction of ovine oPGHS-1, we combined spectroscopic and directed mutagenesis data with X-ray crystallographic refinement of the heme site. Optical and Raman spectroscopy of oxidized oPGHS-1 indicate that its heme iron (Fe(3+)) exists exclusively as a high-spin, six-coordinate species in the holoenzyme and in heme-reconstituted apoenzyme. The sixth ligand is most likely water. The cyanide complex of oxidized oPGHS-1 has a six-coordinate, low-spin ferric iron with a v[Fe-CN] frequency at 445 cm(-)(1); a monotonic sensitivity to cyanide isotopomers that indicates the Fe-CN adduct has a linear geometry. The ferrous iron in reduced oPGHS-1 adopts a high-spin, five-coordinate state that is converted to a six-coordinate, low-spin geometry by CO. The low-frequency Raman spectrum of reduced oPGHS-1 reveals two v[Fe-His] frequencies at 206 and 222 cm(-)(1). These vibrations, which disappear upon addition of CO, are consistent with a neutral histidine (His388) as the proximal heme ligand. The refined crystal structure shows that there is a water molecule located between His388 and Tyr504 that can hydrogen bond to both residues. However, substitution of Tyr504 with alanine yields a mutant having 46% of the peroxidase activity of native oPGHS-1, establishing that bonding of Tyr504 to this water is not critical for catalysis. Collectively, our results show that the proximal histidine ligand in oPGHS-1 is electrostatically neutral. Thus, in contrast to most other peroxidases, a strongly basic proximal ligand is not necessary for peroxidase catalysis by oPGHS-1.
Seibold
S A
SA
Department of Biochemistry, Department of Chemistry and the LASER Laboratory, Michigan State University, East Lansing, Michigan 48824, USA.
Cerda
J F
JF
Mulichak
A M
AM
Song
I
I
Garavito
R M
RM
Arakawa
T
T
Smith
W L
WL
Babcock
G T
GT
D000818:Animals; D018360:Crystallography, X-Ray; D051545:Cyclooxygenase 1; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D014867:Water / Q000737:chemistry
D007527:Isoenzymes; D014867:Water; D006418:Heme; D006639:Histidine; D007501:Iron; D010544:Peroxidases; D051545:Cyclooxygenase 1; D011451:Prostaglandin-Endoperoxide Synthases