Yeast Cytochrome Research Articles

Binding of yeast and human cytochrome c to cardiolipin nanodiscs at physiological ionic strength

Binding of cytochrome c (Cytc) to membranes containing cardiolipin (CL) is of considerable interest because of the importance of this interaction in the early stages of apoptosis. The molecular-level determinants of this interaction are still not well defined and there appear to be species-specific differences in Cytc affinity for CL-containing membranes. Many studies are carried out at low ionic strength far from the 100–150 mM ionic strength within mitochondria. Similarly, most binding studies are done at Cytc concentrations of 10 μM or less, much lower that the estimated range of 0.1 to 5 mM Cytc present in mitochondria. In this study, we evaluate binding of human and yeast Cytc to CL nanodiscs using size exclusion chromatography at 25 μM Cytc concentration and 100 mM ionic strength. We find that yeast Cytc affinity for CL nanodiscs is much stronger than that of human Cytc. Mutational analysis of the site A binding surface shows that lysines 86 and 87 are more important for yeast Cytc binding to CL nanodiscs than lysines 72 and 73, counter to results at lower ionic strength. Analysis of the electrostatic surface potential of human versus yeast Cytc shows that the positive potential due to lysines 86 and 87 and other nearby lysines (4, 5, 11, 89) is stronger than that due to lysines 72 and 73. In the case of human Cytc the positive potential around site A is less uniform and likely weakens electrostatic binding to CL membranes through site A.

Identification of factors limiting the allotopic production of the Cox2 subunit of yeast cytochrome c oxidase.

Mitochondrial genes can be artificially relocalized in the nuclear genome in a process known as allotopic expression, such is the case of the mitochondrial cox2 gene, encoding subunit II of cytochrome c oxidase (CcO). In yeast, cox2 can be allotopically expressed and is able to restore respiratory growth of a cox2-null mutant if the Cox2 subunit carries the W56R substitution within the first transmembrane stretch. However, the COX2W56R strain exhibits reduced growth rates and lower steady-state CcO levels when compared to wild-type yeast. Here, we investigated the impact of overexpressing selected candidate genes predicted to enhance internalization of the allotopic Cox2W56R precursor into mitochondria. The overproduction of Cox20, Oxa1, and Pse1 facilitated Cox2W56R precursor internalization, improving the respiratory growth of the COX2W56R strain. Overproducing TIM22 components had a limited effect on Cox2W56R import, while overproducing TIM23-related components showed a negative effect. We further explored the role of the Mgr2 subunit within the TIM23 translocator in the import process by deleting and overexpressing the MGR2 gene. Our findings indicate that Mgr2 is instrumental in modulating the TIM23 translocon to correctly sort Cox2W56R. We propose a biogenesis pathway followed by the allotopically produced Cox2 subunit based on the participation of the 2 different structural/functional forms of the TIM23 translocon, TIM23MOTOR and TIM23SORT, that must follow a concerted and sequential mode of action to insert Cox2W56R into the inner mitochondrial membrane in the correct Nout-Cout topology.

Assessing the Functional and Structural Stability of the Met80Ala Mutant of Cytochrome c in Dimethylsulfoxide.

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c.

Structural and Functional Investigation of the Interaction between Yeast Cytochrome bc1 Complex and Cytochrome c

Structural and Functional Investigation of the Interaction between Yeast Cytochrome bc1 Complex and Cytochrome c

Search for Novel Lead Inhibitors of Yeast Cytochrome bc1, from Drugbank and COCONUT.

In this work we introduce a novel filtering and molecular modeling pipeline based on a fingerprint and descriptor similarity procedure, coupled with molecular docking and molecular dynamics (MD), to select potential novel quoinone outside inhibitors (QoI) of cytochrome bc1 with the aim of determining the same or different chromophores to usual. The study was carried out using the yeast cytochrome bc1 complex with its docked ligand (stigmatellin), using all the fungicides from FRAC code C3 mode of action, 8617 Drugbank compounds and 401,624 COCONUT compounds. The introduced drug repurposing pipeline consists of compound similarity with C3 fungicides and molecular docking (MD) simulations with final QM/MM binding energy determination, while aiming for potential novel chromophores and perserving at least an amide (R1HN(C=O)R2) or ester functional group of almost all up to date C3 fungicides. 3D descriptors used for a similarity test were based on the 280 most stable Padel descriptors. Hit compounds that passed fingerprint and 3D descriptor similarity condition and had either an amide or an ester group were submitted to docking where they further had to satisfy both Chemscore fitness and specific conformation constraints. This rigorous selection resulted in a very limited number of candidates that were forwarded to MD simulations and QM/MM binding affinity estimations by the ORCA DFT program. In this final step, stringent criteria based on (a) sufficiently high frequency of H-bonds; (b) high interaction energy between protein and ligand through the whole MD trajectory; and (c) high enough QM/MM binding energy scores were applied to further filter candidate inhibitors. This elaborate search pipeline led finaly to four Drugbank synthetic lead compounds (DrugBank) and seven natural (COCONUT database) lead compounds—tentative new inhibitors of cytochrome bc1. These eleven lead compounds were additionally validated through a comparison of MM/PBSA free binding energy for new leads against those obtatined for 19 QoIs.

NMR structural analysis of the yeast cytochrome c oxidase subunit Cox13 and its interaction with ATP

BackgroundMitochondrial respiration is organized in a series of enzyme complexes in turn forming dynamic supercomplexes. In Saccharomyces cerevisiae (baker’s yeast), Cox13 (CoxVIa in mammals) is a conserved peripheral subunit of Complex IV (cytochrome c oxidase, CytcO), localized at the interface of dimeric bovine CytcO, which has been implicated in the regulation of the complex.ResultsHere, we report the solution NMR structure of Cox13, which forms a dimer in detergent micelles. Each Cox13 monomer has three short helices (SH), corresponding to disordered regions in X-ray or cryo-EM structures of homologous proteins. Dimer formation is mainly induced by hydrophobic interactions between the transmembrane (TM) helix of each monomer. Furthermore, an analysis of chemical shift changes upon addition of ATP revealed that ATP binds at a conserved region of the C terminus with considerable conformational flexibility.ConclusionsTogether with functional analysis of purified CytcO, we suggest that this ATP interaction is inhibitory of catalytic activity. Our results shed light on the structural flexibility of an important subunit of yeast CytcO and provide structure-based insight into how ATP could regulate mitochondrial respiration.

Lipid Membrane Interaction of Peptide/DNA Complexes Designed for Gene Delivery.

Among gene delivery systems, peptide-based gene carriers have received significant attention because of their selectivity, biocompatibility, and biodegradability. Since cellular membranes function as a barrier toward exogenous molecules, cell-penetrating peptides (CPPs), which are usually cationic and/or amphiphilic, can serve as efficient carriers to deliver cargo into the cytosol. Here, we examined the interactions of carrier peptides and their DNA complexes with lipid membranes using a quartz crystal microbalance (QCM) and high-speed atomic force microscopy (HS-AFM). The carrier peptides are a 12-residue partial presequence of yeast cytochrome c oxidase subunit IV (Cytcox) and BP100, which are a mitochondria-targeting signal peptide and a CPP, respectively. QCM data showed that BP100 has a higher binding affinity than Cytcox to both plasma membrane- and mitochondrial membrane-mimicking lipid bilayers. The DNA complexes with either Cytcox or BP100 exhibited the same tendency. Furthermore, HS-AFM data demonstrated that the DNA complexes of either peptide can disrupt the lipid membranes, forming larger pores in the case of Cytcox. Our results suggest that the binding affinity of the peptide/DNA complex to the plasma membrane is more critical than its membrane disruption ability in enhancing the cellular uptake of DNA.

A human SCO2 mutation helps define the role of Sco1p in the cytochrome oxidase assembly pathway.

Deficiencies in cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, are most often caused by an inability to complete assembly of the enzyme. Pathogenic mutations in SCO2, which encodes a cytochrome oxidase assembly factor, were recently described in several cases of fatal infantile cardioencephalomyopathy. To determine the molecular etiology of these disorders, we describe the generation and characterization of the parallel mutations in the homologous yeast SCO1 gene. We show that the E155K yeast sco1 mutant is respiration-competent, whereas the S240F mutant is not. Interestingly, the S240F mutation allows partial but incorrect assembly of cytochrome oxidase, as judged by an altered cytochrome aa(3) peak. Immunoblot analysis reveals a specific absence of subunit 2 from the cytochrome oxidase in this mutant. Taken together, our data suggest that Sco1p provides copper to the Cu(A) site on subunit 2 at a step occurring late in the assembly pathway. This is the first instance of a yeast cytochrome oxidase assembly mutant that is partially assembled. The S240F mutant also represents a powerful new tool with which to elucidate further steps in the cytochrome oxidase assembly pathway.

Adsorbing surface strongly influences the pseudoperoxidase and nitrite reductase activity of electrode-bound yeast cytochrome c. The effect of hydrophobic immobilization

The Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) and their adducts with cardiolipin immobilized onto a gold electrode coated with a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol were studied through cyclic voltammetry and surface-enhanced resonance Raman spectroscopy (SERRS). The electroactive species - containing a six-coordinate His/His axially ligated heme and a five-coordinate His/- heme stable in the oxidized and reduced state, respectively - and the pseudoperoxidase activity match those found previously for the wt species and are only slightly affected by CL binding. Most importantly, the reduced His/- ligated form of these variants is able to catalytically reduce the nitrite ion, while electrode-immobilized wt ycc and other His/Met heme ligated variants under a variety of conditions are not. Besides the pseudoperoxidase and nitrite reductase functions, which are the most physiologically relevant abilities of these constructs, also axial heme ligation and the equilibria between conformers are strongly affected by the nature - hydrophobic vs. electrostatic - of the non-covalent interactions determining protein immobilization. Also affected are the catalytic activity changes induced by a given mutation as well as those due to partial unfolding due to CL binding. It follows that under the same solution conditions the structural and functional properties of immobilized ycc are surface-specific and therefore cannot be transferred from an immobilized system to another involving different interfacial protein-SAM interactions.

Modular assembly of yeast mitochondrial ATP synthase and cytochrome oxidase

The respiratory pathway of mitochondria is composed of four electron transfer complexes and the ATP synthase. In this article, we review evidence from studies of Saccharomyces cerevisiae that both ATP synthase and cytochrome oxidase (COX) are assembled from independent modules that correspond to structurally and functionally identifiable components of each complex. Biogenesis of the respiratory chain requires a coordinate and balanced expression of gene products that become partner subunits of the same complex, but are encoded in the two physically separated genomes. Current evidence indicates that synthesis of two key mitochondrial encoded subunits of ATP synthase is regulated by the F1 module. Expression of COX1 that codes for a subunit of the COX catalytic core is also regulated by a mechanism that restricts synthesis of this subunit to the availability of a nuclear-encoded translational activator. The respiratory chain must maintain a fixed stoichiometry of the component enzyme complexes during cell growth. We propose that high-molecular-weight complexes composed of Cox6, a subunit of COX, and of the Atp9 subunit of ATP synthase play a key role in establishing the ratio of the two complexes during their assembly.

Effects of Humanlike Mutations on Yeast Iso-1-Cytochrome C

Apoptosis is a key process in various human diseases. It is initiated in the mitochondria, where changes in the local environment cause cytochrome c (cytc) to become competent for peroxidase activity. Human cytc has 20- to 30-fold lower peroxidase activity than yeast cytc, which we hypothesize allows for tighter control of apoptosis in higher eukaryotes. Many residues that have evolved between yeast and human are found in Ω-loops C and D, which are some of the most dynamic regions of cytc. The Ω-loops are thought to control the dynamics of opening the heme crevice, and thus peroxidase activity. Residues present in human are larger and more hydrophobic than in yeast cytochrome c, so mutations have been made in yeast to reflect that. By mutating residues S40, N63, V57, and A81I, which are larger and more hydrophobic in human cytc, both individually and pairwise in yeast iso-1-cytc, we expect to see corresponding changes in peroxidase activity with minimal changes in overall structure. Circular dichroism was used to monitor guanidine unfolding and assess changes in global stability between each of the variants. The alkaline transition was monitored to estimate the effect of these mutations on Ω-loop D stability. Peroxidase activity measurements were conducted using stopped-flow with guaiacol as a substrate. Our results indicate that single mutations usually have small effects, however in some instances double mutations have more pronounced effects than either single mutation alone. These results seem to indicate that the combined effects of these residues form a network that modulates the peroxidase activity in human cytochrome c, as opposed to one single residue being mainly responsible for these differences.

Mutational analysis of the Qi-site proton pathway in yeast cytochrome bc1 complex

The respiratory cytochrome bc1 complex functions as a protonmotive ubiquinol:cytochrome c oxidoreductase. Lysine 228 (K228) located within the quinol reduction (Qi) site of the bc1 complex, has been reported as a key residue for proton transfer during the redox chemistry cycle to substrate quinone at Qi. In yeast, while single mutations had no effect, the combination of K228L and F225L resulted in a severe respiratory growth defect and inhibition of O2 consumption in intact cells. The inhibition was overcome by uncoupling the mitochondrial membrane or by suppressor mutations in the region of K228L-F225L. We propose that the K228L mutation introduces energetic (and kinetic) barriers into normal electron- and proton transfer chemistry at Qi, which are relieved by dissipation of the opposing protonmotive force or through the restoration of favourable intraprotein proton transfer networks via suppressor mutation.

Quantifying Heme-Protein Maturation from Ratiometric Fluorescence Lifetime Measurements on the Single Fluorophore in Its GFP Fusion.

Protein maturation by heme insertion is a common post-translation modification of key biological importance. Nonetheless, where and when this maturation occurs in eukaryotic cells remain unknown for most heme proteins. Here, we demonstrate for the first time that the maturation of a chromosomally expressed, endogenous heme protein fused to a green fluorescent protein (GFP) can be tracked in live cells. Selecting yeast cytochrome c peroxidase (Ccp1) as our model heme-binding protein, we first characterized the emission in vitro of recombinant Ccp1-GFP with GFP fused C-terminally to Ccp1 by the linker GRRIPGLIN. Time-correlated single-photon counting reveals a single fluorescence lifetime for heme-free apoCcp1-GFP, τ0 = 2.84 ± 0.01 ns. Heme bound to Ccp1 only partially quenches GFP fluorescence since holoCcp1-GFP exhibits two lifetimes, τ1 = 0.95 ± 0.02 and τ2 = 2.46 ± 0.03 ns with fractional amplitudes a1 = 38 ± 1.5% and a2 = 62 ± 1.5%. Also, τ and a are independent of Ccp1-GFP concentration and solution pH between 5.5 and 8.0, and a standard plot of a1 vs % holoCcp1-GFP in mixtures with apoCcp1-GFP is linear, establishing that the fraction of Ccp1-GFP with heme bound can be determined from a1. Fluorescence lifetime imaging microscopy (FLIM) of live yeast cells chromosomally expressing the same Ccp1-GFP fusion revealed 30% holoCcp1-GFP (i.e., mature Ccp1) and 70% apoCcp1-GFP in agreement with biochemical measurements on cell lysates. Thus, ratiometric fluorescence lifetime measurements offer promise for probing heme-protein maturation in live cells, and we can dispense with the reference fluorophore required for ratiometric intensity-based measurements.

Met80 and Tyr67 affect the chemical unfolding of yeast cytochromec: comparing the solutionvs.immobilized state

The motional regime affects the unfolding propensity and axial heme coordination of the Met80Ala and Met80Ala/Tyr67Ala variants of yeast iso-1 cytochromec.

The H channel is not a proton transfer path in yeast cytochrome c oxidase

Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site-directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electrostatic interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.

Calixarene capture of partially unfolded cytochrome c.

Supramolecular receptors such as water-soluble calixarenes are in development as 'molecular glues' for protein assembly. Here, we obtained cocrystals of sulfonato-calix[6]arene (sclx6 ) and yeast cytochrome c (cytc) in the presence of imidazole. A crystal structure at 2.65Å resolution reveals major structural rearrangement and disorder in imidazole-bound cytc. The largest protein-calixarene interface involves 440Å2 of the protein surface with key contacts at Arg13, Lys73, and Lys79. These lysines participate in alkaline transitions of cytc and are part of Ω-loop D, which is substantially restructured in the complex with sclx6 . The structural modification also includes Ω-loop C, which is disordered (residues 41-55 inclusive). These results suggest the possibility of using supramolecular scaffolds to trap partially disordered proteins.

Phosphatidylethanolamine made in the inner mitochondrial membrane is essential for yeast cytochrome bc1 complex function

Of the four separate PE biosynthetic pathways in eukaryotes, one occurs in the mitochondrial inner membrane (IM) and is executed by phosphatidylserine decarboxylase (Psd1). Deletion of Psd1 is lethal in mice and compromises mitochondrial function. We hypothesize that this reflects inefficient import of non-mitochondrial PE into the IM. Here, we test this by re-wiring PE metabolism in yeast by re-directing Psd1 to the outer mitochondrial membrane or the endomembrane system and show that PE can cross the IMS in both directions. Nonetheless, PE synthesis in the IM is critical for cytochrome bc1 complex (III) function and mutations predicted to disrupt a conserved PE-binding site in the complex III subunit, Qcr7, impair complex III activity similar to PSD1 deletion. Collectively, these data challenge the current dogma of PE trafficking and demonstrate that PE made in the IM by Psd1 support the intrinsic functionality of complex III.

Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1.

Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae, CIV is found solely in a SC with cytochrome bc1 (complex III, CIII). Here, we present the cryo-EM structure of S. cerevisiae CIV in a III2IV2 SC at 3.3 Å resolution. While overall similarity to mammalian homologues is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A – involved in allosteric inhibition by ATP – may arise from its association with CIII. The CIII–CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation.

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules

Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix to the intermembrane space. In the yeast Saccharomyces cerevisiae, COX is composed of 11-13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates. Analysis of radiolabeled Cox2p obtained in pulldown assays by native gel electrophoresis revealed the existence of several assembly intermediates, the largest of which had an estimated mass of 450-550 kDa. None of the other known subunits of COX were present in these Cox2p intermediates. This was also true for the several ancillary factors having still undefined functions in COX assembly. In agreement with earlier evidence, Cox18p and Cox20p, previously shown to be involved in processing and in membrane insertion of the Cox2p precursor, were found to be associated with the two largest Cox2p intermediates. A small fraction of the Cox2p module contained Sco1p and Coa6p, which have been implicated in metalation of the binuclear copper site on this subunit. Our results indicate that following its insertion into the mitochondrial inner membrane, Cox2p assembles as a stand-alone protein with the compositionally more complex Cox1p and Cox3p modules.

Structural and functional characterization of the interaction of yeast cytochrome bc1 complex with recombinant cytochrome c

Structural and functional characterization of the interaction of yeast cytochrome bc1 complex with recombinant cytochrome c