Abstract
Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His6-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, kcat, compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.
Highlights
Haemoproteins are an important class of proteins which have diverse biological functions, participating in cellular processes such as respiration, oxygen metabolism and oxygen binding
Recombinant ferrochelatase of the cyanobacterium Synechocystis 6803 was expressed in E. coli with an N-terminal His6-tag (His-FeCh) in inclusion bodies, and no soluble enzyme could be detected after immunoblotting (Fig. 2C, lane 2)
In this work we present detailed enzymological analyses on a functional refolded monomeric type II ferrochelatase from a photosynthetic organism
Summary
Haemoproteins are an important class of proteins which have diverse biological functions, participating in cellular processes such as respiration, oxygen metabolism and oxygen binding. They are often highly represented and the genome of the annual plant Arabidopsis thaliana, for example, encodes as many as ,400 haemoproteins (Arabidopsis Genome Initiative, 2000). Heme is synthesized in a multistep pathway, 5-aminolevulinic acid (ALA) being the earliest precursor. In plants ALA is used to form tetrapyrroles, which – beside heme production - can be used in three different pathways, leading to the production of phytochromobilin, the chromophore of the phytochrome family of red/far-red photoreceptors, to sirohaem, the cofactor of nitrite and sulphite reductases and to chlorophyll (Chl), the pigment responsible for harvesting and trapping light during photosynthesis [1,2]. The terminal enzyme of the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), catalyzing the insertion of ferrous iron into protoporphyrin IX
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