Reaction intermediates in the heme degradation reaction by HutZ from Vibrio cholerae

Abstract

HutZ is a heme-degrading enzyme in Vibrio cholerae. It converts heme to biliverdin via verdoheme, suggesting that it follows the same reaction mechanism as that of mammalian heme oxygenase. However, none of the key intermediates have been identified. In this study, we applied steady-state and time-resolved UV-vis absorption and resonance Raman spectroscopy to study the reaction of heme-HutZ complex with H2O2 or ascorbic acid. We characterized three intermediates: oxyferrous heme, meso-hydroxyheme, and verdoheme complexes. Our data suport the view that HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence and structural homology.

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

“…7. For all Arg92 mutants, absorption of the Soret band decreased rapidly with an increase in absorption at 645-648 nm, suggestive of verdoheme formation [33], as observed at pH 8.0 ( Fig. 5).…”

“…Arg92 appeared necessary for heme degradation when ascorbic acid was used as the electron donor, since almost no iron was released from heme after reaction with ascorbic acid. Considering that the mutants converted heme to verdoheme in the reaction with hydrogen peroxide (H 2 O 2 ), Arg92 may play a key role in transferring H + to a heme degradation intermediate, Fe 3+ -OO -, presumed to be a rate-limiting step of heme degradation by HutZ [33]. In contrast to hHO, HutZ is a dimeric protein.…”

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

“…We further recently succeeded in obtaining the Fe--O 2 stretching mode (ν Fe--O2 ) for heme--HutZ using a rapid mixing technique combined with resonance Raman spectroscopy. 26 The frequency of ν Fe--O2 for oxyferrous heme--HutZ was 565 cm -1 -significantly lower than that for P450 and nitric oxide synthase with Cys as a heme axial ligand (~520 cm -1 ), 38,39 but close to that for proteins with His as a heme axial ligand (~565 cm -1 for hemoglobin). 40,41 The fact that ν Fe--O2 for oxyferrous heme--HutZ was almost the same as that for HO 42 indicated that electron donation from the proximal His of HutZ was at the same level as that of HO, but not as strong as P450 or nitric oxide synthase.…”

“…The Soret band diminished immediately after the addition of H 2 O 2 with a concomitant increase in a band at 644 nm, indicating the formation of verdoheme, as was the case for heme--WT HutZ. Although the band at 644 nm, which is derived from verdoheme, 26 appeared, the absorbance at 644 nm in the reaction of the heme--A31V mutant with H 2 O 2 was approximately 70% of that of heme--WT (Fig. 9C).…”

“…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.…”