Heme Proximal Hydrogen Bonding between His170 and Asp132 Plays an Essential Role in the Heme Degradation Reaction of HutZ from Vibrio cholerae

Abstract

HutZ from Vibrio cholerae is an enzyme that catalyzes the oxygen-dependent degradation of heme. The crystal structure of the homologous protein from Helicobacter pylori, 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 HO 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 (K) 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 K 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.

Subunit–subunit interactions play a key role in the heme-degradation reaction of HutZ fromVibrio cholerae

“…To confirm this, site--directed mutagenesis of Asp132 had previously been performed to reduce electron donation from His170. 37 Unexpectedly, replacement of Asp132 with Asn or Leu was shown to result in a decrease in heme--degradation activity, whereas replacement with Glu, which preserved the proximal hydrogen bond between His170 and Glu132, had little effect on this activity. This result suggests that the hydrogen bond between His170 and Asp132 is not a predominant factor in repressing the heme--degradation activity of HutZ.…”

“…We previously observed the heme--degradation reaction under the single--turnover condition. 25,26,31,37 We here conducted the reaction in the presence of 2 equivalents of heme. When the reaction was initiated by the addition of HutZ to the solution containing 2 equivalent of heme, ascorbic acid and catalase, the heme--degradation ratio, defined as the ratio of iron concentration captured by ferrozine to the initial protein concentration, was ~160% ( Fig.…”

Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

“…The structure of apo-HutZ was solved and its docking structure of heme predicted that Arg92 and His63 coordinate heme [18]. However, the results of our mutational studies suggested that His170 is a sole heme ligand, which was not coincident with the docking model [19,20]. The crystal structure of a HutZ homologous protein HugZ from Helicobacter pylori suggests that His170 of HutZ is the proximal heme ligand, which is within hydrogen bonding distance of Asp132 (Fig.…”

“…The hydrogen bond between Asp132 and His170 could contribute to the imidazolate characteristic of proximal His170, as observed for most peroxidases [21][22][23]. To validate the importance of the proximal hydrogen bond in the heme degradation reaction, Asp132 or His170 was substituted in a previous study by our group [20].…”

Fluorescence Responses of the Protonation and Deprotonation Processes between Phenolate and Phenol within Rosamine

“…[5] In addition to pH detection, the researches of protonated and deprotonated processes give a chance to view the structure-property relationship for functional dyes deeply. [6,7] Hydrogen bond plays an important role not only in the life process [8][9][10] but also in the construction of molecular systems. [11][12][13] Studies have shown that the formation of hydrogen bonds can inhibit the rotation between molecules, which can effectively immobilize the molecular structure, [14] and not only have an impact on the material melting point, boiling point, solubility, viscosity and other properties, [15] but also contribute to the nonradiative energy transitions of the excited state molecules, thus increasing the radiation transition.…”