Electron Transfer between Cytochrome c and the Isolated CuA Domain: Identification of Substrate-Binding Residues in Cytochrome c Oxidase

Abstract

Subunit II of cytochrome c oxidase has a C-terminal domain that is exposed to aqueous solution on membrane surface and contains a copper center called CuA. The central part of the cytochrome c binding site is thought to reside in this domain. We have expressed the subunit II fragment of the Paracoccus denitrificans cytochrome c oxidase in a soluble form and studied its interaction with cytochrome c by stopped-flow spectroscopy. The oxidation of cytochrome c by the CuA domain follows monophasic kinetics, indicating the presence of a single kinetically competent binding site. In low ionic strength medium, the domain oxidizes Paracoccus cytochrome c-550 and horse mitochondrial cytochrome c at the rates of 1.5 x 10(6) and 3 x 10(5) M-1 s-1, respectively. The reaction rates are strongly dependent on ionic strength, which must reflect electrostatic interactions within the complex. The KD for the complex between the bacterial cytochrome c and the domain is 1.6 microM; i.e., it is similar to that between the mitochondrial cytochrome c and the intact oxidase, suggesting that both contain the same catalytically competent binding site. Using site-directed mutagenesis, we have identified five conserved residues of the CuA domain that are involved in the cytochrome c binding. Mutations of glutamine 148, glutamate 154, aspartate 206, aspartate 221, or glutamate 246 lead to a 35-85% decrease in the rate of cytochrome c oxidation. The simultaneous substitution of three invariant carboxylic acids (aspartate 206, aspartate 221, and glutamate 246) leads to a 95% decrease in the reaction rate. Conversely, the reaction can be enhanced by removing a positive charge (lysine 219) from the CuA domain.

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

“…By extrapolating the absolute bimolecular rate constant for the Pd-c 552 -to-Cu A electron transfer couple to 20°C, one obtains a rate of 4.5*10 6 M Ϫ1 s Ϫ1 , and it is possible to compare this to previously reported rate constants for the reaction of the Cu A domain with either cytochrome c 550 from P. denitrificans or horse heart cytochrome c (8). This analysis shows that the reaction with Pd-c 552 is about 3-fold faster than the reaction with Pd-c 550 (1.46*10 6 M Ϫ1 s Ϫ1 ) and 15-fold faster than the reaction with horse heart cytochrome c (3*10 5 M Ϫ1 s Ϫ1 ).…”

“…A soluble cytochrome c 550 is believed to function as an electron donor in different respiratory pathways such as in methanol and methylamine oxidation or in denitrification (7). The kinetics of the electron transfer reaction between this cytochrome and a soluble Cu A domain from P. denitrificans aa 3 cytochrome-c oxidase have been studied (8). However, there is ample evidence that a different cytochrome, cytochrome c 552 , is the genuine mediator between the bc 1 complex and aa 3 .…”

Spectroscopic and Mutagenesis Studies on the CuA Centre from The Cytochrome‐c Oxidase Complex of Paracoccus Denitrificans

“…Additionally, the reduction characteristics of the wild-type Cu, centre have been studied in the isolated Cu,-binding domain (Lappalainen et al, 1995) where it has been shown unequivocally that only one electron is required to reduce the 'as The mutagenesis experiments clearly establish H209, H252, Cys244, and Cys248 as metal ligands. Met255 plays a mainly structural role, but it cannot be excluded as a weaker ligand.…”

Acetyl-L-carnitine physical-chemical, metabolic, and therapeutic properties: relevance for its mode of action in Alzheimer's disease and geriatric depression

“…56 When the positive charge on Lys 219 (exposed to aqueous solution on the membrane surface) of the C-terminal domain of cytochrome C oxidase is removed, the reaction rate is increased. 57 ␣Ketoglutarate dihydrogenase contains three lysines in the lipoyl domain and Lys 43 is the lysine that becomes lipoylated, located at the tip of an exposed ␤-turn. Acetylcholinesterase 58 and choline acetyltransferase 59 also contain lysines.…”