Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae

Abstract

The respiratory supercomplex factors (Rcf) 1 and 2 mediate supramolecular interactions between mitochondrial complexes III (ubiquinolcytochrome c reductase; cyt. bc 1 ) and IV (cytochrome c oxidase; CytcO). In addition, removal of these polypeptides results in decreased activity of CytcO, but not of cyt. bc 1 . In the present study, we have investigated the kinetics of ligand binding, the singleturnover reaction of CytcO with O 2 , and the linked cyt. bc 1 -CytcO quinol oxidation-oxygen-reduction activities in mitochondria in which Rcf1 or Rcf2 were removed genetically (strains rcf1Δ and rcf2Δ, respectively). The data show that in the rcf1Δ and rcf2Δ strains, in a significant fraction of the population, ligand binding occurs over a time scale that is ∼100-fold faster (τ ≅ 100 μs) than observed with the wild-type mitochondria (τ ≅ 10 ms), indicating structural changes. This effect is specific to removal of Rcf and not dissociation of the cyt. bc 1 -CytcO supercomplex. Furthermore, in the rcf1Δ and rcf2Δ strains, the single-turnover reaction of CytcO with O 2 was incomplete. This observation indicates that the lower activity of CytcO is caused by a fraction of inactive CytcO rather than decreased CytcO activity of the entire population. Furthermore, the data suggest that the Rcf1 polypeptide mediates formation of an electrontransfer bridge from cyt. bc 1 to CytcO via a tightly bound cyt. c. We discuss the significance of the proposed regulatory mechanism of Rcf1 and Rcf2 in the context of supramolecular interactions between cyt. bc 1 and CytcO.cytochrome c oxidase | electron transfer | membrane protein | cytochrome aa 3 | cytochrome bc 1

Modulation of O2 reduction in Saccharomyces cerevisiae mitochondria

“…This interaction is proposed to alter the enzymatic properties of the Cyt c O , but the effect is not understood at a molecular level. In a recent study, we showed that the removal of the Rcf1 protein results in two populations of Cyt c O: one that is inactive and one that is essentially fully active . However, in our earlier study, we could not point to the site that was inactivated by removal of Rcf1.…”

Isolation of yeast complex IV in native lipid nanodiscs

“…As observed previously [26] and shown in Figure 4A, also with the mitochondrial membranes we observed a small (~10% at 445 nm) rapid component with a time constant of ~100 s (as well as a major kinetic component with a time constant of ~9 ms). This observation indicates that the altered structural state reflected by the faster CO recombination is present also in the native membranes, although at a lower relative concentration compared to that observed with the CytcO-SMA native nanodiscs.…”

“…Thus, with the detergent-purified CytcO, the rapid component represents CO recombination to heme a 3 in a local structural environment that differs from that in the other two samples (SMA-extracted nanodiscs and native membranes, where the CytcO resides in a membrane). Results from an earlier study with mitochondria showed that a rapid component with a similar time constant of ~100 s and the same spectral features as in detergent-purified CytcO (absorbance decrease at 430 nm) was observed upon removal of the Rcf1 protein [26]. Also this observation indicates that the 100-s component observed with the detergent-purified CytcO reflects CO binding to a…”

“…bc 1 complex. In a recent study we showed that removal of the regulatory Rcf1 protein results in significant changes in the reaction of the reduced CytcO with O 2 [26] and it is therefore important to make sure that this protein copurifies with the CytcO upon isolation of the enzyme. When using "weak" detergents such as e.g.…”

Mutational Analysis of the QRRQ Motif in the Yeast Hig1 Type 2 Protein Rcf1 Reveals a Regulatory Role for the Cytochrome c Oxidase Complex

“…in stoichiometrically equivalent amounts as other complex IV subunits), Rcf1 may exert an influence over substrate binding and the enzymatic properties of complex IV. Indeed, the binding of a Hig1 family member, HIGD1A, to the complex IV enzyme has been indicated to enhance the activity of the enzyme (33), and evidence to indicate altered cytochrome c binding properties to the complex IV enzyme is obtained in mitochondria lacking the Rcf1 protein (15).…”