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

Abstract

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.

The Transcriptomic and Bioinformatic Characterizations of Iron Acquisition and Heme Utilization in Avibacterium paragallinarum in Response to Iron-Starvation

“…Despite extensive studies on the mechanism of heme transport from outside to the cytoplasm, proteins that participated in heme utilization remain unknown. Nowadays, Hut (heme utilization) protein has been identified in Vibrio cholerae according to genome sequence and bioinformatics-based predictions (Uchida et al, 2019). Uchida et al have recently purified and characterized HutZ as a heme-degrading enzyme in V. cholerae, which releases iron into the cytoplasm (Uchida et al, 2012).…”

“…Uchida et al have recently purified and characterized HutZ as a heme-degrading enzyme in V. cholerae, which releases iron into the cytoplasm (Uchida et al, 2012). As a unique heme storage protein, HutZ is critical for heme utilization and can cleave heme to biliverdin via verdoheme (Uchida et al, 2019). Besides, it is reported that HutZ is also required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida (Shi et al, 2019).…”

Metal Sensing by a Glycine–Histidine Repeat Sequence Regulates the Heme Degradation Activity of PM0042 from Pasteurella multocida

“…6 Consequently, reduction of heme, the first step of heme degradation, is slower at pH values >8.0. 3,23 At lower pH, these interactions are weakened and the reduced heme yield is significantly increased.…”

“…Interestingly, the heme degradation rate was increased ∼threefold in the presence of Fe 2+ only (Figure ). Vc HutZ displays pH dependence of heme degradation activity, which is derived from the changes in imidazolate characteristics of the proximal histidine. , Asp132, a highly conserved residue in the HugZ family, forms a hydrogen bond with His170, whose strength is a determinant of the enzymatic activity of Vc HutZ. , In the case of strong interactions between His170 and Asp132, His170 acquires more imidazolate characteristics, which shifts the redox potential of heme iron to a negative value owing to the preference for the oxidized form . Consequently, reduction of heme, the first step of heme degradation, is slower at pH values >8.0. , At lower pH, these interactions are weakened and the reduced heme yield is significantly increased.…”

“…Vc HutZ displays pH dependence of heme degradation activity, which is derived from the changes in imidazolate characteristics of the proximal histidine. , Asp132, a highly conserved residue in the HugZ family, forms a hydrogen bond with His170, whose strength is a determinant of the enzymatic activity of Vc HutZ. , In the case of strong interactions between His170 and Asp132, His170 acquires more imidazolate characteristics, which shifts the redox potential of heme iron to a negative value owing to the preference for the oxidized form . Consequently, reduction of heme, the first step of heme degradation, is slower at pH values >8.0. , At lower pH, these interactions are weakened and the reduced heme yield is significantly increased.…”