Refolding and enzyme kinetic studies on the ferrochelatase of the cyanobacterium synechocystis sp. PCC 6803
2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 2, e55569- p.Article in journal (Refereed) Published
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 220.127.116.11), 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.
Place, publisher, year, edition, pages
2013. Vol. 8, no 2, e55569- p.
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:umu:diva-59788DOI: 10.1371/journal.pone.0055569OAI: oai:DiVA.org:umu-59788DiVA: diva2:556618
The authors are thankful to the Royal Swedish Academy (to C.F.) and the Kempe foundation (to P.S.) for granting their positions. The work was supported by the Swedish Energy Agency and Umea° University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.