ZmPUMP Encodes a Fully Functional Monocot Plant Uncoupling Mitochondrial Protein whose Affinity to Fatty Acid is Increased with the Introduction of a His Pair at the Second Matrix Loop.
Fávaro R.D.1; Borecký J.2; Colombi D.1 and Maia I.G.1
1Instituto de Biociências – Departamento de Genética – UNESP – Botucatu – SP - Brazil; 2Laboratório de Bioenergética, Faculdade de Ciências Médicas (NMCE), UNICAMP – Campinas – SP - Brazil.
The mitochondrial uncoupling protein (UCP) is a specialized transporter protein that uncouples respiration from ATP synthesis. In this study, the cDNA encoding the maize mitochondrial uncoupling protein (ZmPUMP) was expressed in Escherichia coli and reconstituted in liposomes. ZmPUMP activity was associated with an H+ efflux induced by linoleic acid (LA) with a Km of 56.36±0.27 μM and Vmax of 66.9 µmol H+.min-1.(mg prot)-1. LA-induced H+ fluxes were sensitive to ATP inhibition with a Ki of 2.61±0.36 mM at pH 7.2. These values are very close to those measured for reconstituted UCPs from dicot species. ZmPUMP was also used as a model molecule to investigate the importance of a histidine pair found in the second matrix loop segment of mammalian UCP1 and absent in its plant counterparts. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism.
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