Evidence accumulated in the last decade has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes (SC) or respirasomes [1]. ln contrast to the random diffusion model, it is believed that Coenzyme a (CoQ) bound to supercomplexes participates in electron transfer by direct substrate channelling from Complex I to Complex III, providing kinetic advantage for electron transport. ln this perspective, we investigated CoQ behaviour in proteoliposomes from a mitochondrial fraction (R4B) enriched in Complexes I and III in the presence of different amounts of phospholipids and CoQ. lt was found that at high lipid/protein ratios the proteoliposomes exhibited NADH-cytochrome c reductase activity superimposable with that calculated from the pool equation describing randon collisions between Complexes I and III [2], whereas at low lipid/protein ratios the activity was higher than expected from pool behaviour. Concomitantly, it was found by blue-native electrophoresis that in the former case Complexes I and III were individual enzyme units embedded in the lipid membrane, whereas in the latter they were associated in the form of supercomplex I1III2. However, when the supercomplex I1III2 was dissociated by detergent (dodecyl maltoside) the overall NADH-cytochrome c activity was decreased, suggesting the occurrence of pool behaviour. These results provide a demonstration that supercomplex I1III2 confers faster and more efficient electron transfer by direct channelling in the CoQ region (Maranzana E., 2013). We then investigated the kinetics of NADH-cytochrome c reductase as a function of CoQ concentration in proteoliposomes at 1:1 lipid:protein ratio (e.9. presence of supercomplexes) and 30:1 lipid:protein ratio (e.g. absence of supercomplexes). Our preliminary results suggest that both samples exhibit Michaelis-Menten kinetics, in apparent contrast with the presence of substrate channelling by supercomplex I1III2 in the 1:1 sample. However, it is reasoned that CoQ bound in the supercomplex I1III2 is in dissociation equilibrium with the CoQ pool [1]. The finding that the apparent Km for CoQ is lower in the 1:1 sample agrees with this interpretation.
KINETIC PROPERTIES OF SUPERCOMPLEX I1III2 RECONSTITUTED IN VITRO
Gaia Tioli;LENAZ, GIORGIO;GENOVA, MARIA LUISA
2014
Abstract
Evidence accumulated in the last decade has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes (SC) or respirasomes [1]. ln contrast to the random diffusion model, it is believed that Coenzyme a (CoQ) bound to supercomplexes participates in electron transfer by direct substrate channelling from Complex I to Complex III, providing kinetic advantage for electron transport. ln this perspective, we investigated CoQ behaviour in proteoliposomes from a mitochondrial fraction (R4B) enriched in Complexes I and III in the presence of different amounts of phospholipids and CoQ. lt was found that at high lipid/protein ratios the proteoliposomes exhibited NADH-cytochrome c reductase activity superimposable with that calculated from the pool equation describing randon collisions between Complexes I and III [2], whereas at low lipid/protein ratios the activity was higher than expected from pool behaviour. Concomitantly, it was found by blue-native electrophoresis that in the former case Complexes I and III were individual enzyme units embedded in the lipid membrane, whereas in the latter they were associated in the form of supercomplex I1III2. However, when the supercomplex I1III2 was dissociated by detergent (dodecyl maltoside) the overall NADH-cytochrome c activity was decreased, suggesting the occurrence of pool behaviour. These results provide a demonstration that supercomplex I1III2 confers faster and more efficient electron transfer by direct channelling in the CoQ region (Maranzana E., 2013). We then investigated the kinetics of NADH-cytochrome c reductase as a function of CoQ concentration in proteoliposomes at 1:1 lipid:protein ratio (e.9. presence of supercomplexes) and 30:1 lipid:protein ratio (e.g. absence of supercomplexes). Our preliminary results suggest that both samples exhibit Michaelis-Menten kinetics, in apparent contrast with the presence of substrate channelling by supercomplex I1III2 in the 1:1 sample. However, it is reasoned that CoQ bound in the supercomplex I1III2 is in dissociation equilibrium with the CoQ pool [1]. The finding that the apparent Km for CoQ is lower in the 1:1 sample agrees with this interpretation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
