In-Cell Enzymology To Probe His–Heme Ligation in Heme Oxygenase Catalysis

Heme oxygenase (HO) is a ubiquitous
enzyme with key roles in inflammation,
cell signaling, heme disposal, and iron acquisition. HO catalyzes
the oxidative conversion of heme to biliverdin (BV) using a conserved
histidine to coordinate the iron atom of bound heme. This His–heme
interaction has been regarded as being essential for enzyme activity,
because His-to-Ala mutants fail to convert heme to biliverdin in vitro. We probed a panel of proximal His mutants of cyanobacterial,
human, and plant HO enzymes using a live-cell activity assay based
on heterologous co-expression in Escherichia coli of each HO mutant and a fluorescent biliverdin biosensor. In contrast
to in vitro studies with purified proteins, we observed
that multiple HO mutants retained significant activity within the
intracellular environment of bacteria. X-ray crystallographic structures
of human HO1 H25R with bound heme and additional functional studies
suggest that HO mutant activity inside these cells does not involve
heme ligation by a proximal amino acid. Our study reveals unexpected
plasticity in the active site binding interactions with heme that
can support HO activity within cells, suggests important contributions
by the surrounding active site environment to HO catalysis, and can
guide efforts to understand the evolution and divergence of HO function.