Nitric oxide binding to the cardiolipin complex of ferric cytochrome C.
Cardiolipin, a phospholipid specific to the mitochondrion, interacts with the small electron transfer heme protein cytochrome c through both electrostatic and hydrophobic interactions. Once in a complex with cardiolipin, cytochrome c has been shown to undergo a conformational change that leads to the rupture of the bond between the heme iron and the intrinsic sulfur ligand of a methionine residue and to enhance the peroxidatic properties of the protein considered important to its apoptotic activity. Here we report that the ferric cytochrome c/cardiolipin complex binds nitric oxide tightly through a multistep process in which the first step is the relatively slow displacement (5 s(-1)) from heme coordination of an intrinsic ligand that replaces methionine in the complex. Nanosecond photolysis of the nitrosyl adduct demonstrated that a fraction of the nitric oxide escapes from the heme pocket and subsequently recombines to the heme in second-order processes (k = 1.8 × 10(6) and 5.5 × 10(5) M(-1) s(-1)) that, under these conditions, were much faster than recombination of the intrinsic ligand with which they compete. Ultrafast (femtosecond) laser photolysis showed that the geminate recombination of nitric oxide to the heme occurred with time constants (τ = 22 and 72 ps) and that ~23% of the photolyzed nitric oxide escaped into the bulk phase. This high value for the escape fraction relative to other heme proteins indicates the open nature of the heme pocket in this complex. These results are summarized in a scheme and are discussed in terms of the possible modulation of the apoptotic activity of cytochrome c by nitric oxide.
School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, C04 3SQ, UK. silkgi@essex.ac.uk
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Silkstone
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Kapetanaki
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Husu
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Vos
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Wilson
D000818:Animals; D002308:Cardiolipins / Q000737:chemistry / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism*; D006736:Horses; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
D002308:Cardiolipins; D009569:Nitric Oxide; D045304:Cytochromes c; D007501:Iron