Spectral and Kinetic Equivalence of Oxidized Cytochrome c Oxidase as Isolated and “Activated” by Reoxidation

The spectral and kinetic characteristics of two oxidized states of bovine heart cytochrome c oxidase (CcO) have been compared. The first is the oxidized state of enzyme isolated in the fast form (O) and the second is the form that is obtained immediately after oxidation of fully reduced CcO with O2 (OH). No observable differences were found between O and states the of of the enzyme and enzyme in the between and in the of the the and of characteristics is that the and the states of and the CcO in the O and The spectral and kinetic characteristics of two oxidized states of bovine heart cytochrome c oxidase (CcO) have been compared. The first is the oxidized state of enzyme isolated in the fast form (O) and the second is the form that is obtained immediately after oxidation of fully reduced CcO with O2 (OH). No observable differences were found between O and states the of of the enzyme and enzyme in the between and in the of the the and of characteristics is that the and the states of and the CcO in the O and cytochrome c oxidase (CcO) cytochrome c reduced of of cytochrome c reduced of of cytochrome c The of O2 is with the of the of The first is the oxidation of c the of the the of The second is the the of the CcO in and of O2 of and in and is in the of c the oxidase between and the the of and O2 is reduced of the with is the the the reduced enzyme with and that in the and in the fully reduced CcO is oxidized oxidized enzyme is reduced two two the in the is with oxidized enzyme in in the of the oxidized form of two of oxidized CcO immediately after oxidation and the the and the O been that the in the is in the state of the the enzyme and the that differences in the and of in the between the and O the in The of oxidized CcO isolated (O) the fully reduced enzyme fully reduced enzyme with O2 two and the oxidized state is of two is with the of two and of reduced enzyme with two states oxidized enzyme that of oxidized oxidase in the O and form The is oxidase that of the form of enzyme fully reduced enzyme is with The enzyme state and in the of oxidase the CcO the state been that the and in the form with the state of CcO is the that enzyme isolated in the of the and The of and the of oxidized enzyme kinetic and the form that the form of the of that enzyme with form the fast enzyme the of the that fast CcO is enzyme in and is in the state that fast CcO is state of oxidase O oxidase isolated is the isolated enzyme in the fast the that the O state the enzyme enzyme with the state is the enzyme immediately after and is form that the O state the of of and the enzyme been that the of is the of the the the of and that the in the state the enzyme the the of CcO have that the of the of the fast CcO in the O states the kinetic that the of in two the the kinetic and spectral characteristics of the fast enzyme in O and states the CcO CcO in is in The and with the of of and in the two were found the The that the of of fast CcO in O and states and heart cytochrome c reduced form and were and were and of and heart CcO isolated the of and were of in the of the of CcO the of The of is of that of CcO the the enzyme the that CcO with and the of the in the second The second in two the is The CcO the fast with of and of the The CcO the with in The CcO and and CcO two The first of CcO with The the CcO and in and were obtained in with of the of oxidized CcO of isolated is in the fast form and is the form of the enzyme The that the state of enzyme is been that the of CcO is and the is that the of enzyme is of the the of the enzyme the that the of the of of the kinetic of fast and CcO have the form of the isolated fast form and in and the and the is in the of the The that the CcO were of The isolated were with cytochrome c the cytochrome the were of with and c of the in of and and that of cytochrome c is the with and with were with and in the of of and of The were and the The CcO in the of fully reduced oxidized CcO of The of CcO in of of oxidized CcO isolated (O) were in of O in with the of and of were obtained with CcO in the O state in the of and of the of in the state fully reduced CcO in the of and in the with the of the the enzyme is oxidized and and reduced the and the of is of O and were in the the The were the the with of The were in that with The and of O form CcO with c and and the were in of The were reduced CcO in the of c and with of the were with The were in the and in and the the the with the in the the The of The of the the of O fully reduced of in the of of in the of reduced enzyme oxidized and in of of with the the of the and the of with the of of in the of of CcO in the were the of with obtained in with of The of with of the in the of the the in the of the and the were The in found with in the The the of is the of the and of and the of of the oxidized two the of reduced state that is the the the of The of and the fully reduced the and the the of the first and second the The that the of between two is the of the The the of the state the of the reduced The of the in is the of the of and of and were the of reduced oxidized CcO reduced with of been the the of reduced and The of and c the of reduced oxidized CcO and cytochrome c The of reduced and were the of the of and and the and The between the of oxidized cytochrome c and reduced the of of the of cytochrome c the of were the and of the fast and CcO in the O and states in with of the and reduced in the of the and the of of fast CcO the of in two the of the O and states The the and of in O and in of of the fast and enzyme in the O and of of the fast the kinetic O is of CcO is in of of the of CcO is The fast and enzyme were reduced in the of The and the of isolated (O) and enzyme and fast CcO in of the of oxidase in CcO fast CcO in of the of oxidase in in is with enzyme the first of is with of oxidase in the O The of the with the of and fully form is is reduced in the of in with of and The of the the spectral is of CcO in the form the of the is The of the of the fast the The the in that the enzyme is the form of two is with the two in the of and the the of the enzyme and the of enzyme in the fast of of CcO in is in is O and The obtained in of the second the the the in the fully oxidized enzyme of The of of were O and and the second the of is fully oxidized the is of the between the of and is reduced and is oxidized the is and the of is observable in is the the of c and is of two with of and and with of and the two of in O and The of that the state is with the in of CcO have been the of oxidase in the been that two that in the the of and were were the of and The in the and the in with of states of the is been that the oxidized the in the the the the between and the of the and of oxidized with the of the the of the reduced state the the and fully reduced state the kinetic of the of and the of The the of the first the and the second is obtained the and the O the state the and differences in the of the of CcO the first and second in the O and of the of the of the the is that of is in the of is the of the of oxidase found that the of CcO the and of and and of and The of O and in after The with and in the of and the and in in the of two the the and were enzyme with is with the of the of the and the and The is oxidized of oxidized enzyme in the O and The of and the O The of and the the oxidase is of O with in the of reduced enzyme with and after The is the in the of of and is is the of in the after of CcO obtained the between and between the of O and The after the fully reduced enzyme oxidized with The O state in the oxidized CcO with have that oxidized enzyme enzyme that been and of is with is enzyme is in the O state been that differences between O and the of states of and is the O and of the differences in spectral the the fast observable differences between O and the enzyme the enzyme in The of were in and fully reduced and that the of in the O is the the of the of the the of O and the of in the state were the of the of the the O and The of the of that with the of the of O and that is in the of between and in two that the of and in the kinetic that the of and the in O and of of and the and of O and The of that the in two have the and in the the of CcO bovine heart enzyme with is the of the the form in and in The the O state of the is fast form is fully reduced in fully in the been of of enzyme with that the differences in the of is the the of the kinetic and spectral of the O and states the fast form of CcO in that the is that the of the second the reduced oxidase isolated cytochrome c oxidase (CcO) cytochrome c reduced of of cytochrome c reduced of of cytochrome c The of O2 is with the of the of The first is the oxidation of c the of the the of The second is the the of the CcO in and of O2 of and in and is in the of c the oxidase between and the the of and O2 is reduced The of the with is the the the reduced enzyme with and that in the and in the fully reduced CcO is oxidized oxidized enzyme is reduced two two the in the is with oxidized enzyme in in the of the oxidized form of two of oxidized CcO immediately after oxidation and the the and the O been that the in the is in the state of the the enzyme and the that differences in the and of in the between the and O the in The of oxidized CcO isolated (O) the fully reduced enzyme fully reduced enzyme with O2 two and the oxidized state is of two is with the of two and of reduced enzyme with two states oxidized enzyme that of oxidized oxidase in the O and form The is oxidase that of the form of enzyme fully reduced enzyme is with The enzyme state and in the of oxidase the CcO the state been that the and in the form with the state of CcO is the that enzyme isolated in the of the and The of and the of oxidized enzyme kinetic and the form that the form The of the of that enzyme with form the fast enzyme the of the that fast CcO is enzyme in and is in the state that fast CcO is state of oxidase The O oxidase isolated is the isolated enzyme in the fast the that the O state the enzyme enzyme with the The state is the enzyme immediately after and is form that the O state the of of and the enzyme been that the of is the of the the the of and that the in the state the enzyme the the of CcO have that the of the of the fast CcO in the O states the kinetic that the of in two the the kinetic and spectral characteristics of the fast enzyme in O and states the CcO CcO in is in The and with the of of and in the two were found the The that the of of fast CcO in O and states and heart cytochrome c reduced form and were and were and of and heart CcO isolated the of and were of in the of the of CcO the of The of is of that of CcO the the enzyme the that CcO with and the of the in the second The second in two the is The CcO the fast with of and of the The CcO the with in The CcO and and CcO two The first of CcO with The the CcO and in and were obtained in with of the of oxidized CcO of isolated is in the fast form and is the form of the enzyme The that the state of enzyme is been that the of CcO is and the is that the of enzyme is of the the of the enzyme the that the of the of of the kinetic of fast and CcO have the form of the isolated fast form and in and the and the is in the of the The that the CcO were of The isolated were with cytochrome c the cytochrome the were of with and c of the in of and and that of cytochrome c is the with and with were with and in the of of and of The were and the The CcO in the of fully reduced oxidized CcO of The of CcO in of of oxidized CcO isolated (O) were in of O in with the of and of were obtained with CcO in the O state in the of and of the of in the state fully reduced CcO in the of and in the with the of the the enzyme is oxidized and and reduced the and the of is of O and were in the the The were the the with of The were in that with The and of O form CcO with c and and the were in of The were reduced CcO in the of c and with of the were with The were in the and in and the the the with the in the the The of The of the the of O fully reduced of in the of of in the of reduced enzyme oxidized and in of of with the the of the and the of with the of of in the of of CcO in the were the of with obtained in with of The of with of the in the of the the in the of the and the were The in found with in the The the of is the of the and of and the of of the oxidized two the of reduced state that is the the the of The of and the fully reduced the and the the of the first and second the The that the of between two is the of the The the of the state the of the reduced The of the in is the of the of and of and were the of reduced oxidized CcO reduced with of been the the of reduced and The of and c the of reduced oxidized CcO and cytochrome c The of reduced and were the of the of and and the and The between the of oxidized cytochrome c and reduced the of of the of cytochrome c the of were the and and heart cytochrome c reduced form and were and were and of and heart CcO isolated the of and were of in the of the of CcO the of The of is of that of CcO the the enzyme the that CcO with and the of the in the second The second in two the is The CcO the fast with of and of the The CcO the with in The CcO and and CcO two The first of CcO with The the CcO and in and were obtained in with of the of oxidized CcO of CcO isolated is in the fast form and is the form of the enzyme The that the state of enzyme is been that the of CcO is and the is that the of enzyme is of the the of the enzyme the that the of the of of the kinetic of fast and CcO have the form of the isolated fast form and in and the and the is in the of the The that the CcO were of The isolated were with cytochrome c the cytochrome the were of with and c of the in of and and that of cytochrome c is the with and with were with and in the of of and of The were and the The CcO in the of fully reduced oxidized CcO of The of CcO in of of oxidized CcO isolated (O) were in of O in with the of and of were obtained with CcO in the O state in the of and of the of in the state fully reduced CcO in the of and in the with the of the the enzyme is oxidized and and reduced the and the of is The of O and were in the the The were the the with of The were in that with The and of O form CcO with c and and the were in of The were reduced CcO in the of c and with of the were with The were in the and in and the the the with the in the the The of The of the the of O fully reduced of in the of of in the of reduced enzyme oxidized and in of The of with the the of the and the of with the of of The in the of of CcO in the were the of with obtained in with of The of with of the in the of the the in the of the and the were The in found with in the The the of is the of the and of and the of of the oxidized two the of reduced state that is the the the of The of and the fully reduced the and the the of the first and second the The that the of between two is the of the The the of the state the of the reduced The of the in is the of the of and The of and were the of reduced oxidized CcO reduced with of been the the of reduced and The of and c the of reduced oxidized CcO and cytochrome c The of reduced and were the of the of and and the and The between the of oxidized cytochrome c and reduced the of of the of cytochrome c the of were the and of the fast and CcO in the O and states in with of the and reduced in the of the and the of of fast CcO the of in two the of the O and states The the and of in O and in the of isolated (O) and enzyme and fast CcO in of the of oxidase in CcO fast CcO in of the of oxidase in in is with enzyme the first of is with of oxidase in the O The of the with the of and fully form is is reduced in the of in with of and The of the the spectral is of CcO in the form the of the is The of the of the fast the The the in that the enzyme is the form of two is with the two in the of and the the of the enzyme and the of enzyme in the fast of of CcO in is in is O and The obtained in of the second the the the in the fully oxidized enzyme of The of of were O and and the second the of is fully oxidized the is of the between the of and is reduced and is oxidized the is and the of is observable in is the the of c and is of two with of and and with of and the two of in O and The of that the state is with the in of CcO have been the of oxidase in the been that two that in the the of and were were the of and The in the and the in with of states of the is been that the oxidized the in the the the the between and the of the and of oxidized with the of the the of the reduced state the the and fully reduced state the kinetic of the of and the of The the of the first the and the second is obtained the and the O the state the and differences in the of the of CcO the first and second in the O and of the of the of the the is that of is in the of is the of the of oxidase found that the of CcO the and of and and of and The of O and in after The with and in the of and the and in in the of two the the and were enzyme with is with the of the of the and the and The is oxidized of oxidized enzyme in the O and The of and the O The of and the the oxidase is of O with in the of reduced enzyme with and after The is the in the of of and is is the of in the after of CcO obtained the between and between the of O and The after the fully reduced enzyme oxidized with The O state in the oxidized CcO with The of the fast and CcO in the O and states in with of the and reduced in the of the and the of of fast CcO the of in two the of the O and states The the and of in O and in is with enzyme the first of is with of oxidase in the O The of the with the of and fully form is is reduced in the of in with of and The of the the spectral is of CcO in the form the of the is The of the of the fast the The the in that the enzyme is the form of two is with the two in the of and the the of the enzyme and the of enzyme in the fast The of of CcO in is in is O and The obtained in The of the second the the the in the fully oxidized enzyme of The of of were O and and the second the of is fully oxidized the is of the between the of and is reduced and is oxidized the is and the of is observable in is the the of c and is of two with of and and with of and the two of in O and The of that the state is with the in of CcO have been the of oxidase in the been that two that in the the of and were were the of and The in the and the in with The of states of the is been that the oxidized the in the the the the between and the of the and of oxidized with the The of the the of the reduced state the the and fully reduced state the kinetic of the of and the of The the of the first the and the second is obtained the and the O the state the and differences in the of the of CcO the first and second in the O and of the of the of the the is that of is in the of is the of the of oxidase found that the of CcO the and of and and of and The of O and in after The with and in the of and the and in in the of two the the and were enzyme with is with the of the of the and the and The is oxidized The is is the of in the after of CcO obtained the between and between the of O and The after the fully reduced enzyme oxidized with The O state in the oxidized CcO with have that oxidized enzyme enzyme that been and of is with is enzyme is in the O state been that differences between O and the of states of and is the O and of the differences in spectral the the fast observable differences between O and the enzyme the enzyme in The of were in and fully reduced and that the of in the O is the the of the of the the of O and the of in the state were the of the of the the O and The of the of that with the of the of O and that is in the of between and in two that the of and in the kinetic that the of and the in O and of of and the and of O and The of that the in two have the and in the the of CcO bovine heart enzyme with is the of the the form in and in The the O state of the is fast form is fully reduced in fully in the been of of enzyme with that the differences in the of is the the of the kinetic and spectral of the O and states the fast form of CcO in that the is that the of the second the reduced oxidase isolated have that oxidized enzyme enzyme that been and of is with is enzyme is in the O state been that differences between O and the of states of and is the O and of the differences in spectral the the fast observable differences between O and the enzyme the enzyme in The of were in and fully reduced and that the of in the O is the the of the of the the of O and the of in the state were the of the of the the O and The of the of that with the of the of O and that is in the of between and in two that the of and in the kinetic that the of and the in O and The of of and the and of O and The of that the in two have the and in the the of CcO bovine heart enzyme with is the of the the form in and in The the O state of the is fast form is fully reduced in fully in the been of of enzyme with that the differences in the of is the the of the The kinetic and spectral of the O and states the fast form of CcO in that the is that the of the second the reduced oxidase isolated the