HEME OXYGENASE (HO-1) - HIS-132 STABILIZES A DISTAL WATER LIGAND AND ASSISTS CATALYSIS

His-25 and His-132 are the primary candidates for the proximal heme ir on ligand in heme oxygenase isozyme-1 (HO-1). The unambiguous spectros copic demonstration that His-25 is the proximal iron ligand leaves the role of His-132 uncertain. Absorption and resonance Raman spectroscop y are used here to establish that mutation of His-132 to an alanine, g lycine, or serine does not alter the histidine-iron bond, but results in the loss of the water molecule coordinated to the distal side of th e iron in the wild-type enzyme-substrate complex. The His-132 mutation s also (a) destabilize the ferrous-O-2 complex with respect to autoxid ation, which should result in partial uncoupling of NADPH consumption from heme oxidation, and (b) decrease the affinity of the enzyme for h eme. The catalytic activity of the protein is decreased but not suppre ssed by these mutations: the H132G and H132A mutants retain 40-50% aci d the H132S mutant 20% of the activity of the wild-type protein, His-1 32, however, is required for catalytic turnover of the protein with H2 O2. These results place His-132 close to the iron on the distal side o f the heme pocket and indicate that His-132 facilitates, but is not ab solutely required for, the catalytic turnover of HO-1.