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Heme Proximal Hydrogen Bonding between His170 and Asp132 Plays an Essential Role in the Heme Degradation Reaction of HutZ from <i>Vibrio cholerae</i>
HutZ from <i>Vibrio cholerae</i> is an enzyme that catalyzes
the oxygen-dependent degradation of heme. The crystal structure of
the homologous protein from <i>Helicobacter pylori</i>,
HugZ, predicts that Asp132 in HutZ is located within hydrogen-bonding
distance of the heme axial ligand His170. Hydrogen bonding between
His170 and Asp132 appears to be disfavored in heme-degrading enzymes,
because it can contribute to the imidazolate character of the axial
histidine, as observed in most heme-containing peroxidases. Thus,
we investigated the role of this potential hydrogen bond in the heme
degradation reaction by mutating Asp132 to Leu, Asn, or Glu and by
mutating His170 to Ala. Heme degradation activity was almost completely
lost in D132L and D132N mutants, whereas verdoheme formation through
reaction with H<sub>2</sub>O<sub>2</sub> was comparable in the D132E
mutant and wild-type enzyme. However, even at pH 6.0, when the heme
is in a high-spin state, the D132E mutant was inactive toward ascorbic
acid because of a significant reduction in its affinity (<i>K</i><sub>d</sub>) for heme (4.1 μM) compared with that at pH 8.0
(0.027 μM). The heme degradation activity of the H170A mutant
was also substantially reduced, although this mutant bound heme with
a <i>K</i><sub>d</sub> of 0.067 μM, despite the absence
of an axial ligand. Thus, this study showed that proximal hydrogen
bonding between Asp132 and His170 plays a role in retaining the heme
in an appropriate position for oxygen-dependent heme degradation