Monday, November 26, 2012

Cytochrome biogenesis work selected in chemical biology Faculty of 1000

A recent paper from work of Drs Mavridou and Stevens in Professor Ferguson’s lab, in collaboration with David Phillips BBSRC fellow Dr Allen, has been selected by the Faculty of 1000 (F1000) as a significant contribution in the field of chemical biology (1).
Published in the Journal of Biological Chemistry, the paper entitled “A pivotal heme-transfer reaction intermediate in cytochrome c biogenesis" provides an insight into the biosynthesis of heme proteins (2). It describes the trapping, identification and characterisation of a long sought-after intermediate in the biogenesis pathway for the heme proteins cytochromes c.
Cytochromes are a ubiquitous and essential class of proteins. They contain heme, a lipophilic moiety with chelated iron and one of the most common iron-containing molecules in nature. Heme is toxic to living cells - it is able to promote the formation of reactive species and has a tendency to polymerise –and is therefore usually found in nature in association with heme-chaperones or proteins that need a heme prosthetic group to carry out their function. Cytochromes perform electron transfer through their heme cofactor.
C-type cytochromes are characterised by the covalent attachment of heme to the polypeptide chain via a conserved CXXCH motif. They are vital to the growth of nearly all types of cells by participating in respiration, photosynthesis and biosynthesis of cofactors, and in eukaryotes are central to signalling apoptosis.
Covalent heme attachment to cytochromes is an essential post-translational modification. Four different protein systems have been identified across organisms that carry out this modification. Professor Ferguson’s laboratory has studied System I, which is found predominantly in Gram-negative bacteria, archaea and plant mitochondria, for some years. Intriguingly, it is the most complex of the maturation systems, containing at least eight proteins.
The central step of heme attachment depends on the membrane-bound heme chaperone CcmE which transfers heme to the cytochrome after itself binding the cofactor via a covalent bond (Figure 1). The membranous nature of this system makes in vivo studies challenging; proteins are expressed at low levels and are prone to degradation and aggregation. The interaction and transfer of heme from the heme chaperone to the cytochrome has been assumed to be the pivotal step of the cytochrome c maturation
pathway for years but this has never been directly proven.
The work from the Ferguson and Allen groups, funded by the BBSRC and Wellcome Trust, has now provided direct and unambiguous evidence that cytochrome acquires its cofactor directly from CcmE. A covalent complex between CcmE, heme and the cytochrome was trapped in vivo for the first time.
The researchers attribute their success to the use of a variant of a b-type cytochrome (cytochrome b562) which can be expressed recombinantly in large quantities. This cytochrome is unique because it is stable in the cell even when it lacks its heme cofactor. As well as successfully trapping the complex, the researchers also found that the quantities of this normally transient intermediate species are responsive in an expected way to mutations in genes for the various cytochrome c maturation proteins - providing a new way of assessing the function of this ensemble of proteins.
The c-type cytochrome biogenesis apparatus occurs in phylogenetically diverse organisms from all kingdoms of life. Identification of the central intermediate of this widely found process provides proof of the pathway for heme provision. Whilst much remains unknown about other aspects of heme biology –such as how it is transported around cells for its myriad of functions –the work demonstrating how heme is transferred from a heme chaperone to a major class of hemoproteins in a widespread post-translational modification pathway is a significant advancement.

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