Thomas Kleinschroth

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc1 complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.

Mgr2 promotes coupling of the mitochondrial presequence translocase to partner complexes

Mgr2 is a new component of the mitochondrial presequence translocase required for efficient coupling of the TIM23 core complex to Tim21, respiratory chain complexes, and the translocase of the outer mitochondrial membrane.

Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE

We previously proposed that the dimeric cytochrome bc1 complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc1 monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289–22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc1 complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10–16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c1 by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.

X-ray structure of the dimeric cytochrome bc1 complex from the soil bacterium Paracoccus denitrificans at 2.7-Å resolution

The respiratory cytochrome bc(1) complex is a fundamental enzyme in biological energy conversion. It couples electron transfer from ubiquinol to cytochrome c with generation of proton motive force which fuels ATP synthesis. The complex from the α-proteobacterium Paracoccus denitrificans, a model for the medically relevant mitochondrial complexes, lacked structural characterization. We show by LILBID mass spectrometry that truncation of the organism-specific, acidic N-terminus of cytochrome c(1) changes the oligomerization state of the enzyme to a dimer. The fully functional complex was crystallized and the X-ray structure determined at 2.7-Å resolution. It has high structural homology to mitochondrial complexes and to the Rhodobacter sphaeroides complex especially for subunits cytochrome b and ISP. Species-specific binding of the inhibitor stigmatellin is noteworthy. Interestingly, cytochrome c(1) shows structural differences to the mitochondrial and even between the two Rhodobacteraceae complexes. The structural diversity in the cytochrome c(1) surface facing the ISP domain indicates low structural constraints on that surface for formation of a productive electron transfer complex. A similar position of the acidic N-terminal domains of cytochrome c(1) and yeast subunit QCR6p is suggested in support of a similar function. A model of the electron transfer complex with membrane-anchored cytochrome c(552), the natural substrate, shows that it can adopt the same orientation as the soluble substrate in the yeast complex. The full structural integrity of the P. denitrificans variant underpins previous mechanistic studies on intermonomer electron transfer and paves the way for using this model system to address open questions of structure/function relationships and inhibitor binding.

Asymmetric Binding of Stigmatellin to the Dimeric Paracoccus denitrificans bc1 Complex EVIDENCE FOR ANTI-COOPERATIVE UBIQUINOL OXIDATION AND COMMUNICATION BETWEEN CENTER P UBIQUINOL OXIDATION SITES

We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc1 complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2–3 nm shift of the visible absorbance spectrum of the bL heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the bL spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the bL spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the bL heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.

The obligate respiratory supercomplex from Actinobacteria

Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (Em(ISP)=+160mV, Em(bL)=-291mV, Em(bH)=-163mV, Em(cc)=+100mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (Em(CuA)=+150mV, Em(a/a3)=+143/+317mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity.

Photoinitiated electron transfer within the Paracoccus denitrificans cytochrome bc1 complex: mobility of the iron-sulfur protein is modulated by the occupant of the Q(o) site.

Domain rotation of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c(1) redox centers plays a key role in the mechanism of the cyt bc(1) complex. Electron transfer within the cyt bc(1) complex of Paracoccus denitrificans was studied using a ruthenium dimer to rapidly photo-oxidize cyt c(1) within 1 μs and initiate the reaction. In the absence of any added quinol or inhibitor of the bc(1) complex at pH 8.0, electron transfer from reduced ISP to cyt c(1) was biphasic with rate constants of k(1f) = 6300 ± 3000 s(-1)and k(1s) = 640 ± 300 s(-1) and amplitudes of 10 ± 3% and 16 ± 4% of the total amount of cyt c(1) photooxidized. Upon addition of any of the P(m) type inhibitors MOA-stilbene, myxothiazol, or azoxystrobin to cyt bc(1) in the absence of quinol, the total amplitude increased 2-fold, consistent with a decrease in redox potential of the ISP. In addition, the relative amplitude of the fast phase increased significantly, consistent with a change in the dynamics of the ISP domain rotation. In contrast, addition of the P(f) type inhibitors JG-144 and famoxadone decreased the rate constant k(1f) by 5-10-fold and increased the amplitude over 2-fold. Addition of quinol substrate in the absence of inhibitors led to a 2-fold increase in the amplitude of the k(1f) phase. The effect of QH(2) on the kinetics of electron transfer from reduced ISP to cyt c(1) was thus similar to that of the P(m) inhibitors and very different from that of the P(f) inhibitors. The current results indicate that the species occupying the Q(o) site has a significant conformational influence on the dynamics of the ISP domain rotation.

Characterization of mutations in crucial residues around the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans

The protonation state of residues around the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site‐directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme bL edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild‐type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Qo site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P. denitrificans as well. The protonated residue E295 is proposed to change the hydrogen‐bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X‐ray structures.

The acidic domain of cytochrome c1 in Paracoccus denitrificans, analogous to the acidic subunits in eukaryotic bc1 complexes, is not involved in the electron transfer reaction to its native substrate cytochrome c552☆

The cytochrome bc(1) complex is a key component in several respiratory pathways. One of the characteristics of the eukaryotic complex is the presence of a small acidic subunit, which is thought to guide the interaction of the complex with its electron acceptor and facilitate electron transfer. Paracoccus denitrificans represents the only example of a prokaryotic organism in which a highly acidic domain is covalently fused to the cytochrome c(1) subunit. In this work, a deletion variant lacking this acidic domain has been produced and purified by affinity chromatography. The complex is fully intact as shown by its X-ray structure, and is a dimer (Kleinschroth et al., subm.) compared to the tetrameric (dimer-of-dimer) state of the wild-type. The variant complex is studied by steady-state kinetics and flash photolysis, showing wild type turnover and a virtually identical interaction with its substrate cytochrome c(552).