Fungal Heme Oxygenases: Functional Expression and Characterization of Hmx1 from Saccharomyces cerevisiae and CaHmx1 from Candida albicans
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- 作者：Donghak Kim ; Erik T. Yukl ; Pierre Moë ; nne-Loccoz ; Paul R. Ortiz de Montellano
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：December 12, 2006
- 年：2006
- 卷：45
- 期：49
- 页码：14772 - 14780
- 全文大小：223K
- 年卷期：v.45,no.49(December 12, 2006)
- ISSN：1520-4995
文摘
Heme oxygenases convert heme to free iron, CO, and biliverdin. Saccharomyces cerevisiaeand Candida albicans express putative heme oxygenases that are required for the acquisition of iron fromheme, a critical process for fungal survival and virulence. The putative heme oxygenases Hmx1 andCaHmx1 from S. cerevisiae and C. albicans, respectively, minus the sequences coding for C-terminalmembrane-binding domains, have been expressed in Escherichia coli. The C-terminal His-tagged, truncatedenzymes are obtained as soluble, active proteins. Purified ferric Hmx1 and CaHmx1 have Soret absorptionmaxima at 404 and 410 nm, respectively. The apparent heme binding Kd values for Hmx1 and CaHmx1are 0.34 ± 0.09 
M and 1.0 ± 0.2 M, respectively. The resonance Raman spectra of Hmx1 reveal aheme binding pocket similar to those of the mammalian and bacterial heme oxygenases. Several reductants,including ascorbate, yeast cytochrome P450 reductase (CPR), human CPR, spinach ferredoxin/ferredoxinreductase, and putidaredoxin/putidaredoxin reductase, are able to provide electrons for biliverdin productionby Hmx1 and CaHmx1. Of these, ascorbate is the most effective reducing partner. Heme oxidation byHmx1 and CaHmx1 regiospecifically produces biliverdin IX
. Spectroscopic analysis of aerobic reactionswith H2O2 identifies verdoheme as a reaction intermediate. Hmx1 and CaHmx1 are the first fungal hemeoxygenases to be heterologously overexpressed and characterized. Their heme degradation activity isconsistent with a role in iron acquisition.
M and 1.0 ± 0.2 M, respectively. The resonance Raman spectra of Hmx1 reveal aheme binding pocket similar to those of the mammalian and bacterial heme oxygenases. Several reductants,including ascorbate, yeast cytochrome P450 reductase (CPR), human CPR, spinach ferredoxin/ferredoxinreductase, and putidaredoxin/putidaredoxin reductase, are able to provide electrons for biliverdin productionby Hmx1 and CaHmx1. Of these, ascorbate is the most effective reducing partner. Heme oxidation byHmx1 and CaHmx1 regiospecifically produces biliverdin IX. Spectroscopic analysis of aerobic reactionswith H2O2 identifies verdoheme as a reaction intermediate. Hmx1 and CaHmx1 are the first fungal hemeoxygenases to be heterologously overexpressed and characterized. Their heme degradation activity isconsistent with a role in iron acquisition.