Architecture of Active Mammalian Respiratory Chain Supercomplexes

Abstract

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals. In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals. All living organisms use a series of integral membrane protein complexes for energy conversion and ATP synthesis. In eukaryotes, electrons are transported by the respiratory chain, starting from NADH via complex I (NADH:ubiquinone from via complex the membrane integral electron complex III the electron and complex IV to the electron chain a proton the inner mitochondrial membrane, which is by complex to synthesize ATP. In the structures of the respiratory chain complexes from organisms for bovine heart mitochondrial complex III and IV A structure of complex I is electron is in organisms and a map from for the bovine heart complex I for the arrangement of the respiratory chain complexes in the membrane According to the of the respiratory chain in the membrane, and electron the of the protein complexes and the electron In the proposed the is from to the supercomplexes of different in and in mitochondria from and by of and In these studies, supercomplexes of of complex I complex III and complex IV in A of these respiratory supercomplexes the of the complexes by in bovine heart mitochondria is for the arrangement of the respiratory chain complexes and their an electron projection map of a supercomplex of complex I and complex III for the architecture of a supercomplex complexes and By the most supercomplexes in bovine heart mitochondria are a supercomplex of complex I and III and supercomplex complexes I and III and IV of and By electron we image supercomplexes from bovine heart mitochondria and complex IV in the and heart mitochondria were in and to and in a of of of the to a of of a of in and were of the and in and for to a of and to protein were by a and were to and by of complex I determined by in to a of and of complex IV by of and in and of complexes I and III were by a from in of complex I by the of NADH a of of complex III determined by the a of to by complex the the and the were the the supercomplexes and were the for and the the were in the and from the to the All were and the and supercomplex were also and were a a of electron image were by for and of and and a a of to the were to a of were the bovine heart mitochondria were a of of of the by protein of respiratory chain complexes in shows the complexes two supercomplex I1III2 of complex I and a dimer of complex and I1III2IV1 complex and supercomplex of a and a the of of protein for of the respiratory chain complexes to I1III2 and I1III2IV1 the two supercomplex a and the of complex which of their In protein were the of the supercomplexes I1III2 and biochemical and supercomplexes I1III2 and I1III2IV1 were from the and of the supercomplexes by an to supercomplex which I1III2IV1 in the supercomplex I1III2 two complexes I and were of supercomplexes by In of the complexes were the and from complex protein from complex and IV from complex IV enzymatic of supercomplex to their NADH of complex I and of complex were by NADH of complex I by from of complex IV determined by the of to and complex I for supercomplexes I1III2 and I1III2IV1, complex IV for supercomplex I1III2IV1 in the were and were to of complex I and of complex III in the the activity, the complex I in supercomplex I1III2 about the of in supercomplex I1III2IV1 III active in supercomplex I1III2IV1, supercomplex I1III2 In supercomplexes activity, supercomplex I1III2IV1 of the supercomplex I1III2 and in a electron the supercomplexes I1III2 and 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supercomplex and to the different a different by the supercomplex fits the of the complex III dimer and IV a which in the complex IV a to the which is the of in the of the the views a and which views to the membrane from the and of the of the the two views are to and By comparing the and views of supercomplex I1III2IV1, the of complex I in the views a the of the a and In the the of the complex I the membrane is in the views a and complex IV in the I1III2IV1 the supercomplex I1III2 the were and to the I1III2IV1 views two of the A difference map of I1III2IV1 I1III2 a of the the complex I closely the projection of a complex IV from the the and and is the complex IV the of the supercomplex the which is the dimer in the x-ray structure the complex IV and the of complex I the membrane of complexes I and were from the of complex III dimer and to were the two in the supercomplex for the in the supercomplex and were the for the were complex from the a the in the supercomplex However, the for the two by location of I and in the and two respiratory chain supercomplexes I1III2 and I1III2IV1 from bovine heart mitochondria were in an active and were by electron the two supercomplexes I1III2 and I1III2IV1 are to I1III2 a of the supercomplex structures we for the two supercomplexes their of I1III2 and I1III2IV1, from complex III a dimer in the structures of the bovine and structures the complex is a which is by the two in their and their and electrons are between in the two a complex III dimer is in the supercomplexes of bovine heart and mitochondria the of a dimer for complex In of structures for the supercomplexes biochemical complex III dimer is for of complex I heart complex IV is also in the structure is in the supercomplex and IV is the of the supercomplex the which is the dimer in the x-ray of complex IV and of a single in the supercomplex a a of dimer dimer in the complex IV is and is by of the and by the of III of the the these the two are by to is dimer supercomplex the 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complexes I and IV a complex IV is to complex in the of complex IV is to complex between the two supercomplexes and from bovine heart mitochondria complex IV to complexes I and However, of the supercomplex I1III2IV1 in a by an the complexes and I1III2IV1, a of the supercomplex biochemical the of complex the in the an arrangement of the complexes proposed in complex IV to complex the location of complex and the membrane of complex I in the a map of the complexes is are the of I1III2 and I1III2IV1 were proposed biochemical In these the supercomplex I1III2IV1 is a major of the respiratory chain in mitochondria. All living organisms use a series of integral membrane protein complexes for energy conversion and ATP synthesis. In eukaryotes, electrons are transported by the respiratory chain, starting from NADH via complex I (NADH:ubiquinone from via complex the membrane integral electron complex III the electron and complex IV to the electron chain a proton the inner mitochondrial membrane, which is by complex to synthesize ATP. 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and III and IV of and By electron we image supercomplexes from bovine heart mitochondria and complex IV in the assembly. and heart mitochondria were in and to and in a of of of the to a of of a of in and were of the and in and for to a of and to protein were by a and were to and by of complex I determined by in to a of and of complex IV by of and in and of complexes I and III were by a from in of complex I by the of NADH a of of complex III determined by the a of to by complex the the and the were the the supercomplexes and were the for and the the were in the and from the to the All were and the and supercomplex were also and were a a of electron image were by for and of and and a a of to the were to a of were and heart mitochondria were in and to and in a of of of the to a of of a of in and were of the and in and for to a of and to protein were by a and were to and by of complex I determined by in to a of and of complex IV by of and in and of complexes I and III were by a from in of complex I by the of NADH a of of complex III determined by the a of to by complex the the and the were the the supercomplexes and were the for and the the were in the and from the to the All were and the and supercomplex were also and were a a of electron image were by for and of and and a a of to the were to a of were the bovine heart mitochondria were a of of of the by protein of respiratory chain complexes in shows the complexes two supercomplex I1III2 of complex I and a dimer of complex and I1III2IV1 complex and supercomplex of a and a the of of protein for of the respiratory chain complexes to I1III2 and I1III2IV1 the two supercomplex a and the of complex which of their In protein were the of the supercomplexes I1III2 and biochemical and supercomplexes I1III2 and I1III2IV1 were from the and of the supercomplexes by an to supercomplex which I1III2IV1 in the supercomplex I1III2 two complexes I and were of supercomplexes by In of the complexes were the and from complex protein from 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supercomplex from the a of the a from the in the are the number of to image projection of the supercomplexes. views from the of supercomplex I1III2IV1 of A of and determined by to the the supercomplex I1III2 of difference map of I1III2IV1 the of of supercomplex I1III2IV1 of the from a the the of the the two views and from the the the the of the the and the are in the views in a and are of the bovine complex IV x-ray structure from the image the views by a of of of supercomplex I1III2IV1 were After and the image two of the the their known electron microscopic and x-ray the in to the complexes I and a projection map of the bovine heart complex I of the of I of by the the of complex membrane of complex I in the supercomplex to the in the supercomplex the of the protein by and from the a the of the of the complexes I and in supercomplex projection structure of bovine complex of of the of I of by are views of x-ray structures of bovine complex IV and bovine complex III dimer the the 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of the A difference map of I1III2IV1 I1III2 a of the the complex I closely the projection of a complex IV from the the and and is the complex IV the of the supercomplex the which is the dimer in the x-ray structure the complex IV and the of complex I the membrane of complexes I and were from the of complex III dimer and to were the two in the supercomplex for the in the supercomplex and were the for the were complex from the a the in the supercomplex However, the for the two by location of I and in the and the bovine heart mitochondria were a of of of the by protein of respiratory chain complexes in shows the complexes two supercomplex I1III2 of complex I and a dimer of complex and I1III2IV1 complex and supercomplex of a and a the of of protein for of the respiratory chain complexes to I1III2 and I1III2IV1 the two supercomplex a and the of complex which of their In protein were the of the supercomplexes I1III2 and biochemical and supercomplexes I1III2 and I1III2IV1 were from the and of the supercomplexes by an to supercomplex which I1III2IV1 in the supercomplex I1III2 two complexes I and were of supercomplexes by In of the complexes were the and from complex protein from complex and IV from complex IV enzymatic of supercomplex to their NADH of complex I and of complex were by NADH of complex I by from of complex IV determined by the of to and complex I for supercomplexes I1III2 and I1III2IV1, complex IV for supercomplex I1III2IV1 in the were and were to of complex I and of complex III in the the activity, the complex I in supercomplex I1III2 about the of in supercomplex I1III2IV1 III active in supercomplex I1III2IV1, supercomplex I1III2 In supercomplexes activity, supercomplex I1III2IV1 electron the supercomplexes I1III2 and I1III2IV1 were of supercomplex the In were in the I1III2 are in the the complex III dimer from of the is the supercomplex I1III2 the is an particle of supercomplex I1III2IV1 were for image and to and of the and were All of different a were the of the and and the and All of the the two views the two of the supercomplexes the to the is the the views by a of of of supercomplex I1III2IV1 were After and the image two of the the their known electron microscopic and x-ray the in to the complexes I and a projection map of the bovine heart complex I of the of I of by the the of complex membrane of complex I in the supercomplex to the in the supercomplex the of the protein by and from the a the of the of complex I of the of I of by a the membrane, is to in the supercomplex and to the different a different by the supercomplex assembly. fits the of the complex III dimer and IV a which in the complex IV a to the which is the of in the of the the views a and which views to the membrane from the and of the of the the two views are to and By comparing the and views of supercomplex I1III2IV1, the of complex I in the views a the of the a and In the the of the complex I the membrane is in the views a and complex IV in the I1III2IV1 the supercomplex I1III2 the were and to the I1III2IV1 views two of the A difference map of I1III2IV1 I1III2 a of the the complex I closely the projection of a complex IV from the the and and is the complex IV the of the supercomplex the which is the dimer in the x-ray structure In the complex IV and the of complex I the membrane of complexes I and were from the of complex III dimer and to were the two in the supercomplex for the in the supercomplex and were the for the were complex from the a the in the supercomplex However, the for the two by location of I and in the and two respiratory chain supercomplexes I1III2 and I1III2IV1 from bovine heart mitochondria were in an active and were by electron the two supercomplexes I1III2 and I1III2IV1 are to I1III2 a of the supercomplex structures we for the two supercomplexes their of I1III2 and I1III2IV1, from complex III a dimer in the structures of the bovine and structures the complex is a which is by the two in their and their and electrons are between in the two a complex III dimer is in the supercomplexes of bovine heart and mitochondria the of a dimer for complex In of structures for the supercomplexes biochemical complex III dimer is for of complex I heart complex IV is also in the structure is in the supercomplex and IV is the of the supercomplex the which is the dimer in the x-ray of complex IV and of a single in the supercomplex a a of dimer dimer in the complex IV is and is by of the and by the of III of the the these the two are by to is dimer supercomplex the of supercomplexes in bovine heart mitochondria, which in the number of complex IV from to the of complex IV is However, the and of I1III2IV1 the of complex IV in supercomplex is After I1III2IV1 and a of I1III2 into its a for complex IV in the of the the supercomplex I1III2 projection map from mitochondria the between complexes I and to different from in bovine heart mitochondrial supercomplexes. bovine supercomplex I1III2 is the of the supercomplex is and bovine complex I in their which the difference between and complex IV and the of complex I of the respiratory chain supercomplex I1III2IV1 were complex I and in the the arrangement of complexes I and in the views of supercomplex I1III2IV1, we two their location determined the electron In complex IV is the and the of complex I the of the supercomplex I1III2IV1 from the and complex is the and the membrane of I the of the complex and IV are to complex I a In the complex is the of the and to complexes I and membrane of complex I is the complexes I and IV a complex IV is to complex in the of complex IV is to complex between the two supercomplexes and from bovine heart mitochondria complex IV to complexes I and However, of the supercomplex I1III2IV1 in a by an the complexes and I1III2IV1, a of the supercomplex biochemical the of complex the in the an arrangement of the complexes proposed in complex IV to complex the location of complex and the membrane of complex I in the a map of the complexes is are the of I1III2 and I1III2IV1 were proposed biochemical In these the supercomplex I1III2IV1 is a major of the respiratory chain in mitochondria. two respiratory chain supercomplexes I1III2 and I1III2IV1 from bovine heart mitochondria were in an active and were by electron the two supercomplexes I1III2 and I1III2IV1 are to I1III2 a of the supercomplex structures we for the two supercomplexes their of I1III2 and I1III2IV1, from complex III a dimer in the structures of the bovine and structures the complex is a which is by the two in their and their and electrons are between in the two a complex III dimer is in the supercomplexes of bovine heart and mitochondria the of a dimer for complex In of structures for the supercomplexes biochemical complex III dimer is for of complex I heart complex IV is also in the structure is in the supercomplex and IV is the of the supercomplex the which is the dimer in the x-ray of complex IV and of a single in the supercomplex a a of dimer 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the membrane of I the of the complex and IV are to complex I a In the complex is the of the and to complexes I and membrane of complex I is the complexes I and IV a In complex IV is to complex in the of complex IV is to complex between the two supercomplexes and from bovine heart mitochondria complex IV to complexes I and However, of the supercomplex I1III2IV1 in a by an the complexes and I1III2IV1, a of the supercomplex biochemical the of complex the in the an arrangement of the complexes proposed in complex IV to complex the location of complex and the membrane of complex I in the a map of the complexes is are the of I1III2 and I1III2IV1 were proposed biochemical In these the supercomplex I1III2IV1 is a major of the respiratory chain in mitochondria. and for the bovine complex I and for electron and for in the and for and of the