Cytochrome B5 from Giardia Lambliaw

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The protozoan intestinal parasite Giardia lamblia lacks mitochondria and the ability to make haem yet encodes several putative haem-binding proteins, including three of the cytochrome b5 family. We cloned one of these (gCYTb5-I) and expressed it within Escherichia coli as a soluble holoprotein. UV-visible and resonance Raman spectra of gCYTb5-I resemble those of microsomal cytochrome b5, and homology modelling supports a structure in which a pair of invariant histidine residues act as axial ligands to the haem iron. The reduction potential of gCYTb5-I is 165 mV vs. SHE and is relatively low compared to most values (110 to +80 mV) for this class of protein. The amino- and carboxy-terminal sequences that flank the central haembinding core of the Giardia cytochromes are highly charged and differ from those of other family members. A core gCYTb5-I variant lacking these flanking sequences was also able to bind haem. The presence of one actual and two probable functional cytochromes b5 in Giardia is evidence of uncharacterized cytochrome-mediated metabolic processes within this medically important protist. Introduction

The protozoan parasite Giardia lamblia is one of the most
common causes of infectious diarrhoea, known as giardiasis.
Infection of the host begins when dormant Giardia cysts are
ingested through contaminated water, food or faecal–oral
contact. Exposure of the cysts to the acidic environment of
the host’s stomach triggers their excystation, and the resulting flagellated trophozoites adhere to the epithelial cells of the upper small intestine where the trophozoites reproduce by
asexual binary fission. As trophozoites pass through the
intestinal tract, they convert to cysts, which are excreted with the faeces to become the source for new infections. Giardiasis occurs worldwide but is a particular concern in underdeveloped regions, and it is a target of the World Health Organization’s Neglected Diseases Initiative.1

Apart from its significance to human and animal health,
Giardia is an interesting organism because of the many unusual features that set it apart from other eukaryotes. These differences in cell structure, gene expression, and metabolism arise not only from its parasitic mode of existence but also from its evolutionary divergence. Certain eukaryotic organelles such as peroxisomes are absent, and Giardia has only a rudimentary Golgi apparatus.

Instead of mitochondria, Giardia possesses mitosomes, organelles descended from mitochondria that retain the ability to assemble iron–sulphur clusters but that lack the capacity for oxidative phosphorylation.2 Transcriptional control of gene expression within Giardia also differs from most eukaryotes, with comparatively short promoters and 50 untranslated regions.3 Giardia is a

microaerotolerant facultative anaerobe that relies on substrate-level phosphorylation rather than oxidative phosphorylation to meet its needs for ATP, and it also uses pyrophosphate as a key energy intermediate.4 As a parasite, Giardia relies on the intestinal milieu of its host for most amino acids and complex lipids, and it lacks enzymes for common metabolic processes such as the citric acid cycle.5

Early biochemical characterization of Giardia overlooked
the possibility that this protist might use haem or haemcontaining proteins. In retrospect, this oversight was based
on over-generalization of several observations, most notably the lack of mitochondrial respiration and catalase activity for which haem is essential.5 This misconception ended with
publication of the draft genome of Giardia in 2007.6 While
Giardia has no recognized genes encoding enzymes required for haem biosynthesis, it does possess several genes that encode putative haem-binding proteins. One of these genes encodes a functional flavohemoglobin with nitric oxide dioxygenase activity;7 expression of this protein is induced in the presence of nitric oxide donor compounds to result in a three-fold increase in...
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