Covalent binding of arylazido derivatives of cytochrome c to cytochrome oxidase

On the function of multiple subunits of cytochrome c oxidase from higher eukaryotes

“…2. Subunit II is assumed to contain heme a since cytochrome c binds specifically to this subunit: (i) Dithiobis(succinimidylpropionate) crosslinked cytochrome c with subunit II when reacted with the cytochrome c-cytochrome c oxidase complex [74]; (ii) Arylazido-derivatives ofcytochrome c reacted with subunit II of beef heart [75,76] and yeast [77] cytochrome c oxidase. 3.…”

Photoaffinity labeling of yeast cytochrome oxidase with arylazido cytochrome c derivatives

“…Preparations of cytochrome c oxidase [lo] from beef-hearts, and of cytochrome c arylazido derivatives [5] [5,6] . Cytochrome c oxidase activity and concentration were measured according to [5].…”

“…In this work yeast cytochrome oxidase was reacted with arylazido cytochrome c in order to compare the subunits of yeast and beef-heart cytochrome oxidase which are crosslinked when this derivative is used [5,6] . Furthermore, subunit II, which may migrate anomalously with respect to subunit III [9] , was identified in our experimental conditions by labeling its highly reactive SHgroup withN-['4C]ethylmaleimide.…”

“…Using horse heart cytochrome c labeled with a 3nitrophenylazido group at lysine 13, evidence was provided [5,6] that subunit II was the subunit nearest to cytochrome c in the complex with the beef-heart oxidase. This conclusion was also supported by [7] in which dithiobissuccininidylpropionate was used as crosslinking reagent.…”

Cytochrome c oxidase from Paracoccus denitrificans: both hemes are located in subunit I.

“…Such a conclusion may be important, since it would mean that subunit II needed to bind only copper and cytochrome c. The cytochrome c binding property has been confirmed by several approaches that indicate that subunit II participates with other subunits to create the cytochrome c binding domain (15)(16)(17) (23) without the addition of polymerized 0.5% polyacrylamide. Slab gels were conventionally stained with Coomassie blue or by silver as described by Bio-Rad.…”