Ionic strength effects on cytochrome aa3 kinetics

Reaction of thionitrobenzoate‐modified yeast cytochrome c with monomeric and dimeric forms of beef heart cytochrome c oxidase

“…Essentially maximal activity was observed for the beef heart enzyme when exogenous cytochrome c was added along with TMPD and ascorbate [6]. This result was taken to support the proposal in [24] that (exogenous) cytochrome c in the low affinity site for substrate increased the off rate of substrate (in this case covalently bound cytochrome c) from the high affinity site (for re-reduction with artificial electron donors in the above experiment). Our findings offer an alternative explanation.…”

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

“…The interaction must 1) facilitate the rapid formation of a highly specific 1:1 complex with CcO, 2) stabilize the optimal orientation of the complex for rapid electron transfer to the initial acceptor in CcO, and 3) allow rapid dissociation of the product complex to release ferricytochrome c. The need for both rapid substrate complex formation and product complex dissociation would appear to be particularly stringent. Steady-state kinetic studies revealed that the reaction rate was limited by product dissociation at low ionic strength, reached an optimum at intermediate ionic strength, and was strongly inhibited at high ionic strength, indicating a significant electrostatic interaction between the two proteins (1)(2)(3)(4)(5)(6). Extensive chemical modification studies have shown that six or seven highly conserved lysine amino groups surrounding the heme crevice of cytochrome c are involved in the electrostatic complex with cytochrome oxidase (7)(8)(9)(10)(11)(12)(13)).…”

Modulation of cytochrome oxidase kinetics by indirect antibody action

“…When the ionic strength is reduced to 5 mM phosphate and the assay is carried out polarographically the control enzyme shows the characteristic biphasic behaviour towards cytochrome c concentration ( fig.2A). This behaviour has been interpreted in terms of 'high-& lowaffinity' binding sites for the substrate [18,19] but it may be noted the phenomenon is also capable of several other types of interpretation [20,21]. Fig.2A shows that upon addition of anti-subunit V, the enzyme is always inhibited, but that the pattern of inhibition differs in the two phases of the plot.…”

“…The alternative assumes that cytochrome c at the low-affinity site increases the reduction rate of tightly bound cytochrome c [23]. In both models product dissociation limits Vm~x at low ionic strengths and low concentrations of cytochrome c [19,23]. However, the turnover at high levels of c (Vmx at the low-affinity site) is largely limited by the speed of internal electron transfer, as it is independent of ionic strength (see [23,24].…”