The two isoforms of human heme oxygenase (HO1 and HO2) catalyze oxidative degradation of heme to biliverdin, Fe, and CO. Unlike HO1, HO2 contains two C-terminal heme regulatory motifs (HRMs) centered at Cys265 and Cys282 that act as redox switches and, in their reduced dithiolate state, bind heme (Fleischhacker et al., Biochemistry, 2015, 54, 2693-2708). Here, we describe cryoreduction/annealing and electron paramagnetic resonance spectroscopic experiments to study the structural features of the oxyheme moiety in HO2 and to elucidate the initial steps in heme degradation. We conclude that the same mechanism of heme hydroxylation to α-meso-hydroxyheme is employed by both isoforms and that the HRMs do not affect the physicochemical properties of the oxy-Fe(II) and HOO-Fe(III) states of HO2. However, the absorption spectrum of oxy-Fe(II)-HO2 is slightly blue-shifted relative to that of HO1. Furthermore, heme hydroxylation proceeds three times more slowly, and the oxy-Fe(II) state is 100-fold less stable in HO2 than in HO1. These distinctions are attributed to slight structural variances in the two proteins, including differences in equilibrium between open versus closed conformations. Kinetic studies revealed that heme oxygenation by HO2 occurs solely at the catalytic core in that a variant of HO2 lacking the C-terminal HRM domain exhibits the same specific activity as one containing both the catalytic core and HRM domain; furthermore, a truncated variant containing only the HRM region binds but cannot oxidize heme. In summary, HO1 and HO2 share similar catalytic mechanisms, and the HRMs do not play a direct role in the HO2 catalytic cycle.
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