Heme degradation reaction of HutZ from Vibrio cholerae is regulated by the subunit–subunit interactions.

HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes degradation of heme. The highly conserved Arg92 residue in the HutZ family is proposed to interact with an iron-bound water molecule in the distal heme pocket. To clarify the specific role of Arg92 in the heme degradation reaction, the residue was substituted with alanine, leucine, histidine or lysine to modulate electrostatic interactions with iron-bound ligand. All four Arg92 mutants reacted with hydrogen peroxide to form verdoheme, a prominent intermediate in the heme degradation process. However, when ascorbic acid was used as an electron source, iron was not released even at pH 6.0 despite a decrease in the Soret band, indicating that non-enzymatic heme degradation occurred. Comparison of the rates of heme reduction, ligand binding and verdoheme formation suggested that proton transfer to the reduced oxyferrous heme, a potential rate-limiting step of heme degradation in HutZ, is hampered by mutation. In our previous study, we found that the increase in the distance between heme and Trp109 from 16 to 18 Å upon lowering the pH from 8.0 to 6.0 leads to activation of ascorbic acid-assisted heme degradation by HutZ. The distance in Arg92 mutants was >19 Å at pH 6.0, suggesting that subunit-subunit interactions at this pH are not suitable for heme degradation, similar to Asp132 and His63 mutants. These results suggest that interactions of Arg92 with heme-bound ligand induce alterations in the distance between subunits, which plays a key role in controlling the heme degradation activity of HutZ.

PM0042 protein from the Gram-negative bacterial pathogen Pasteurella multocida is homologous to the heme-degrading enzyme HutZ belonging to the pyridoxine-5-phosphate oxidase-like family. A characteristic feature of PM0042 is possession of a glycine−histidine (GH) repeat sequence at the C-terminal region. In this study, we examined the heme degradation ability of PM0042, with a particular focus on the role of the GH repeat sequence. PM0042 was expressed in Escherichia coli and successfully purified using a nickel (Ni 2+ )-affinity column without a histidine tag, suggesting that its GH motif facilitates binding to Ni 2+ . Reaction with ascorbic acid induced a significant decrease in the Soret band, suggesting the breakage of heme. While a Fe 2+ −ferrozine complex was not formed upon addition of ferrozine to the solution after the reaction, prior addition of metal ions to fill the metal binding site in the GH repeat sequence led to increased complex formation. In the presence of Fe 2+ , the heme degradation rate was accelerated ∼threefold, supporting the theory that Fe 2+ binds the PM0042 protein (possibly at the GH repeat sequence) and enhances its heme degradation activity. In contrast to HutZ from Vibrio cholerae in which enzymatic activity is regulated by the protonation status of the heme proximal ligand, heme reduction is not the rate-determining step for PM0042. Rather, proton transfer to reduced oxyheme is affected, as established with the H 2 O/D 2 O isotope experiment. Based on the collective findings, the GH repeat sequence of PM0042 is proposed to function as a metal sensor that modulates iron uptake via the heme-degrading process in P. multocida.