Mutations in the tether region of the iron–sulfur protein affect the activity and assembly of the cytochrome bc1 complex of yeast mitochondria

Abstract

Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.

Structure and Function of CytochromebcComplexes

“…Inhibition by the disulfide linking could be by preventing the helix from extending, so that the "back end" of the extrinsic domain is held close to the membrane and the cluster cannot pivot into position. However, this interpretation seems inconsistent with results from several labs in the bacterial (44,238) and yeast (239) systems, which show that the neck can be shortened by three (44,239) or even five (238) residues and still function. The authors attributed inhibition by the disulfide bridge to an increase in rigidity of the neck region.…”

“…Suppressor strains that correct mutations in the C-terminal part of the ISP or in cyt b through mutation in ISP have been interpreted as showing an importance of the hinge region (233)(234)(235), and in several studies, the hinge region has been modified directly. These include extensive substitution studies, including changes in length of the hinge region (44,(236)(237)(238)(239)(240)(241). In some convincing experiments by Tian et al (237), cysteines have been substituted in pairs at the helical repeat along the sequence that undergoes extension.…”

“…The authors attributed inhibition by the disulfide bridge to an increase in rigidity of the neck region. While shortening of the neck region is tolerated, extension by even one residue is deleterious, and by more than one, strongly inhibitory (44,238,239). Two possible explanations come to mind.…”

Functional Insensitivity of the Cytochrome b6f Complex to Structure Changes in the Hinge Region of the Rieske Iron-Sulfur Protein

“…that introduction of excess flexibility by substitution with 6 Gly residues (19) or of extra length by insertion of 1 or more residues (17,24,26) in the hinge region is deleterious to the function of the bc 1 complex (Table III, parts A and B). With 4 -7 glycine residues in the sequence, the ISP hinge region in the b 6 f complex is predicted to be structure-less and more flexible than that in the bc 1 complex (Table I).…”

“…It was subsequently found that the function of the complex is very sensitive to perturbation of the sequence and structure of the hinge region by sitedirected mutagenesis in which the hinge was altered by residue deletion, insertion, and substitution (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Although these data were interpreted in terms of a requirement for the mobility of the ISP soluble domain, the extreme sensitivity of bc 1 function to increases in hinge flexibility (19) and length (17,24,26) cannot be predicted or inferred from the x-ray structure data. Thus, it is of interest to study the function of the putative hinge region in the b 6 f complex (Table I).…”