Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex.

Mitochondrial cytochrome bc1 complex performs two functions: It is a respiratory multienzyme complex and it recognizes a mitochondrial targeting presequence. Refined crystal structures of the 11-subunit bc1 complex from bovine heart reveal full views of this bifunctional enzyme. The "Rieske" iron-sulfur protein subunit shows significant conformational changes in different crystal forms, suggesting a new electron transport mechanism of the enzyme. The mitochondrial targeting presequence of the "Rieske" protein (subunit 9) is lodged between the two "core" subunits at the matrix side of the complex. These "core" subunits are related to the matrix processing peptidase, and the structure unveils how mitochondrial targeting presequences are recognized.

Structure of a water soluble fragment of the 'Rieske' iron-sulfur protein of the bovine heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5 A resolution.

BACKGROUND: The 'Rieske' iron-sulfur protein is the primary electron acceptor during hydroquinone oxidation in cytochrome bc complexes. The spectroscopic and electrochemical properties of the 'Rieske' [2Fe-2S] cluster differ significantly from those of other iron-sulfur clusters. A 129-residue water soluble fragment containing the intact [2Fe-2S] cluster was isolated following proteolytic digestion of the bc1 complex and used for structural studies. RESULTS: The structure of the Rieske iron-sulfur fragment containing the reduced [2Fe-2S] cluster has been determined using the multiwavelength anomalous diffraction (MAD) technique and refined at 1.5 A resolution. The fragment has a novel overall fold that includes three sheets of beta strands. The iron atoms of the [2Fe-2S] cluster are coordinated by two cysteine (Fe-1) and two histidine (Fe-2) residues, respectively, with the histidine ligands completely exposed to the solvent. This is in contrast to the four cysteine coordination pattern observed in previously characterised [2Fe-2S] ferredoxins. The cluster-binding fold is formed by two loops connected by a disulfide bridge; these loops superpose with the metal-binding loops of rubredoxins. The environment of the cluster is stabilised by an extensive hydrogen-bond network. CONCLUSIONS: The high-resolution structure supports the proposed coordination pattern involving histidine ligands and provides a basis for a detailed analysis of the spectroscopic and electrochemical properties. As the cluster is located at the tip of the protein, it might come into close contact with cytochrome b. The exposed N epsilon atoms of the histidine ligands of the cluster are readily accessible to quinones and inhibitors within the hydroquinone oxidation (QP) pocket of the bc1 complex and may undergo redox-dependent protonation/deprotonation.

Role of the mitochondrial bc1-complex in the cytotoxic action of diethylstilbestrol-diphosphate toward prostatic carcinoma cells.

In previous work (P. Schulz et al., Cancer Res., 48: 2867-2870, 1988) we have demonstrated that diethylstilbestrol (DES), DES-monophosphate, and DES-diphosphate (DESDP) are generally cytotoxic at concentrations attained in patients' sera during therapeutic DESDP infusions for progressed carcinoma of the prostate. We have extended this work and addressed two questions: (a) Is DESDP itself a completely nontoxic prodrug which has to be transformed into the active species DES by a phosphatase? (b) Which metabolic or regulatory mechanism in a cell is the target of DES action? Using cell cultures in phosphatase-depleted media we could provide evidence that DESDP exerts cytotoxic activity only after conversion to DES. Oxygen electrode experiments and difference spectra with intact mitochondria demonstrated that DES did not act as an uncoupler, but inhibited electron flow from ubiquinone to cytochrome c1. Phenomena previously observed in DES-treated cells could be explained by distortion of the energy metabolism.

Functional implications of the structure of the 'Rieske' iron-sulfur protein of bovine heart mitochondrial cytochrome bc1 complex.

Recently, we have determined the structure of the catalytic domain of the 'Rieske' iron-sulfur protein of bovine heart mitochondrial bc1 complex at 1.5 A resolution (Iwata, S., Saynovits, M., Link, T.A. and Michel, H. (1996) Structure, 4, 567-579). This is the first structure of a bis-histidine coordinated [2Fe-2S] cluster. The spectroscopic, electrochemical, and functional implications of the structure will be discussed.

Structural and functional analysis of deficient mutants in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae.

Four point mutations in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae that had been selected for respiratory incompetence but still contained spectrally detectable haem aa3 were analysed. The isolated mutant enzymes exhibited minor band shifts in their optical spectra and contained all eleven subunits. However, steady state activities were only a few percent compared to wild type enzyme. Using a comprehensive experimental approach, we first checked the integrity of the enzyme preparations and then identified the specific functional defect. The results are discussed using information from the recently solved structures of cytochrome c oxidase at 2.8 A. Mutation 167N is positioned between haem a and a conserved glutamate residue (E243). It caused a distortion of the EPR signal of haem a and shifted its midpoint potential by 54 mV to the negative. The high-resolution structure suggests that the primary reason for the low activity of the mutant enzyme could be that asparagine in position 67 might form a stable hydrogen bond to E243, which is part of a proposed proton channel. Cytochrome c oxidase isolated from mutant T316K did not meet our criteria for homogeneity and was therefore omitted from further analysis. Mutants G352V and V380M exhibited an impairment of electron transfer from haem a to a3 and ligand binding to the binuclear centre was affected. In mutant V380M also the midpoint potential of CuB was shifted by 65 mV to the positive. The results indicated for these two mutants changes primarily associated with the binuclear centre, possibly associated with an interference in the routes and/or sites of protonation which are required for stable formation of the catalytic intermediates. This interpretation is discussed in the light of the high resolution structure.

Biochemical and spectroscopic properties of the four-subunit quinol oxidase (cytochrome ba3) from Paracoccus denitrificans.

The ba3 quinol oxidase from Paracoccus denitrificans has been purified by a new protocol leading to significantly higher yields than previously reported (Richter et al. (1994) J. Biol. Chem. 269, 23079-23086). In an SDS PAG an additional protein band compared with the previous preparation appears, which can be identified as the major form of subunit II. All protein bands can be assigned to genes of the qox operon by N-terminal sequencing, indicating that the oxidase consists of four subunits. In addition to one heme A, one heme B, and one copper atom, the preparation contains two ubiquinone molecules per enzyme. The oxidase is further characterized by electron paramagnetic resonance (EPR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy.

MRS3 and MRS4, two suppressors of mtRNA splicing defects in yeast, are new members of the mitochondrial carrier family.

When present in high copy number plasmids, the nuclear genes MRS3 and MRS4 from Saccharomyces cerevisiae can suppress the mitochondrial RNA splicing defects of several mit- intron mutations. Both genes code for closely related proteins of about Mr 32,000; they are 73% identical. Sequence comparisons indicate that MRS3 and MRS4 may be related to the family of mitochondrial carrier proteins. Support for this notion comes from a structural analysis of these proteins. Like the ADP/ATP carrier protein (AAC), the mitochondrial phosphate carrier protein (PiC) and the uncoupling protein (UCP), the two MRS proteins have a tripartite structure; each of the three repeats consists of two hydrophobic domains that are flanked by specific amino acid residues. The spacing of these specific residues is identical in all domains of all proteins of the family, whereas spacing between the hydrophobic domains is variable. Like the AAC protein, the MRS3 and MRS4 proteins are imported into mitochondria in vitro and without proteolytic cleavage of a presequence and they are located in the inner mitochondrial membrane. In vivo studies support this mitochondrial localization of the MRS proteins. Overexpression of the MRS3 and MRS4 proteins causes a temperature-dependent petite phenotype; this is consistent with a mitochondrial function of these proteins. Disruption of these genes affected neither mitochondrial functions nor cellular viability. Their products thus have no essential function for mitochondrial biogenesis or for whole yeast cells that could not be taken over by other gene products. The findings are discussed in relation to possible functions of the MRS proteins in mitochondrial solute translocation and RNA splicing.

The role of the 'Rieske' iron sulfur protein in the hydroquinone oxidation (Q(P)) site of the cytochrome bc1 complex. The 'proton-gated affinity change' mechanism.

The essential reaction in the widely accepted proton-motive Q-cycle mechanism of the bc1 complex is the bifurcation of the electron flow during hydroquinone oxidation at the hydroquinone oxidation (Q(P)) site formed by the 'Rieske' iron sulfur protein and by the heme bL domain of cytochrome b. The 'Rieske' [2Fe-2S] cluster has a unique structure containing two exposed histidine ligands, which are the binding site for quinones. The affinity of the 'Rieske' cluster for quinones increases several orders of magnitude upon reduction; this will stabilize semiquinone at the Q(P) site. Based on this affinity change, a reaction scheme is presented which can explain the bifurcation of the electron flow without invoking highly unstable semiquinone species.

Analysis of the structures of the subunits of the cytochrome bc1 complex from beef heart mitochondria.

The interaction of the protein subunits of the bc1 complex from beef heart is analysed on the basis of protein chemical data and of secondary structure predictions suggesting a large number of amphipathic helices. Electrostatic interactions, i.e. helix-dipole interactions and ionic bonds, may play a major role in the stabilisation of the arrangement of the subunits within the multi-protein complex, formation of subcomplexes and maintenance of the steric strain of cytochrome b. A model of the heme-carrying 'core' of cytochrome b, i.e. of helices II-V, is presented consisting of a twisted '4-alpha-helical' bundle held together by helix-dipole interactions and stabilised by the interaction with other protein subunits of the bc1 complex.

Solute carriers involved in energy transfer of mitochondria form a homologous protein family.

The sequences of three mitochondrial carriers involved in energy transfer, the ADP/ATP carrier, phosphate carrier and uncoupling carrier, are analyzed. Similarly to what has been previously reported for the ADP/ATP carrier and the uncoupling protein, now also the phosphate carrier is found to have a tripartite structure comprising three similar repeats of approx. 100 residues each. The three sequences show a fair overall homology with each other. More significant homologies are found by comparing the repeats within and between the carriers in a scheme where the sequences are spliced into repeats, which are arranged for maximum homology by allowing possible insertions or deletions. A striking conservation of critical residues, glycine, proline, of charged and of aromatic residues is found throughout all nine repeats. This is indicative of a similar structural principle in the repeats. Hydropathy profiles of the three proteins and a search for amphipathic alpha-spans reveal six membrane-spanning segments for each carrier, providing further support for the basic structural identity of the repeats. The proposed folding pattern of the carriers in the membrane is exemplified with the phosphate carrier. A possible tertiary arrangement of the repeats and the membrane-spanning helices is shown. The emergence of a mitochondrial carrier family by triplication and by divergent evolution from a common gene of about 100 residues is discussed.

Electrochemical study of the redox properties of [2Fe-2S] ferredoxins. Evidence for superreduction of the Rieske [2Fe-2S] cluster.

Direct, unmediated electrochemistry has been used to compare the redox properties of [2Fe-2S] clusters in spinach ferredoxin, Spirulina platensis ferredoxin and the water soluble fragment of the Rieske protein. The use of electrochemistry enabled, for the first time, the observation of the second reduction step, [Fe(III), Fe(II)] to [Fe(II), Fe(II)], in a biological [2Fe-2S] system. A water-soluble fragment of the Rieske protein from bovine heart bc1 complex exhibits two subsequent quasi-reversible responses in cyclic voltammetry on activated glassy carbon. In contrast the ferredoxins from spinach and Spirulina platensis only show one single reduction potential. These results support a seniority scheme for biological iron-sulfur clusters related cluster size to electron transfer versatility. Electrochemical reduction of spinach ferredoxin in the presence of NADP+ and ferredoxin: NADP+ oxidoreductase results in the generation of NADPH. The second order rate constant for the reaction between the ferredoxin and the reductase was estimated from cyclic voltammetry experiments to be > 3.10(5) M-1.s-1.

Isolation and amino acid sequence of the 8 kDa DCCD-binding protein of beef heart ubiquinol:cytochrome c reductase.

The 8 kDa protein of beef heart ubiquinol:cytochrome c reductase was detected by means of a new SDS-PAGE [(1985) FEBS Lett. 190, 89-94] system and was isolated by a series of chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence was determined by solid-phase Edman degradation of both the N-terminal part of the whole protein and proteolytic cleavage fragments of the protein. The protein consists of 78 amino acid residues: its Mr was calculated to be 7998. Structure predictions have been made from average and sided hydropathy profiles. The suggested structure encompasses an alpha-helix and a beta-strand, the latter comprising a glutamic acid residue situated in a relatively hydrophobic neighbourhood. This residue may be responsible for the fact that the 8 kDa protein is the first subunit of the whole reductase (consisting of 11 subunits) to be labelled by DCCD when the reductase is in free form or inlaid in phospholipid vesicles.

Isolation and amino acid sequence of the smallest subunit of beef heart bc1 complex.

The 11 subunits of beef heart bc1 complex can be separated either by a new SDS-PAGE system or by a series of chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence of the smallest subunit was determined by complete solid-phase Edman degradation and was confirmed by sequencing the N-terminal part and the C-terminal tryptic fragment by liquid-phase Edman degradation. The protein consists of 56 amino acid residues; the Mr was calculated to be 6363. The protein ('ISP binding factor') might be entangled in the reassembly of the iron-sulfur protein with the bc1 subcomplex.

Isolation and amino acid sequence of the 'Rieske' iron sulfur protein of beef heart ubiquinol:cytochrome c reductase.

The sequence of the 'Rieske' iron sulfur protein from the bc1 complex of beef heart mitochondria has been determined by solid phase Edman degradation of the whole protein and of various proteolytic fragments. The protein consists of 196 amino acid residues. The molecular mass of the apoprotein was calculated to be 21,536 Da, that of the holo-protein including the Fe2S2 cluster as 21,708 Da. The protein is mainly hydrophilic with a polarity index of 42.9% and 25% of charged residues. It contains a hydrophobic membrane anchor which is predicted to form a 'hairpin' structure. The iron sulfur cluster is bound near the C-terminus of the protein between a hydrophobic and a more amphipathic domain. This reflects the fact that the cluster is located near the outer surface of the inner mitochondrial membrane. A folding pattern describing all known features of the protein is proposed.

Isolation and amino acid sequence of the 9.5 kDa protein of beef heart ubiquinol:cytochrome c reductase.

The 9.5 kDa protein of beef heart ubiquinol:cytochrome c reductase was isolated by a series of chromatographic steps involving dissociation of the complex by urea and guanidine. A clear distinction between the 9.5 kDa protein and the 9.2 kDa protein described earlier [(1982) J. Biochem. 91, 2077-2085] by SDS-PAGE was only achieved when the electrophoresis was performed according to Schägger et al. [(1985) FEBS Lett. 190, 89-94; (1986) Methods Enzymol. 126, 22] because in this gel system the apparent molecular mass of the 9.5 kDa protein is shifted to 11 kDa. The amino acid sequence was determined by solid-phase Edman degradation of the whole protein up to amino acid residue 80 and of the proteolytic cleavage fragments. The protein consists of 81 amino acid residues; its Mr was calculated to be 9507. Structure predictions have been made from average and sided hydropathy profiles. The 9.5 kDa protein is either bound to the core proteins within a 9.5 kDa-core protein subcomplex or else it aggregates easily with the core proteins during the isolation procedure.

A comparative picosecond-resolved fluorescence study of tryptophan residues in iron-sulfur proteins.

The fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission from six Fe/S proteins (ranging from the very simplest ones to enzyme complexes containing one, two or more Trp residues) were measured. All proteins were examined in the reduced and the oxidized state. In either redox state each protein exhibits ultrarapid tryptophan fluorescence decay on the picosecond timescale contributing up to 93% of the total decay. Correlation times in the range of 1 ns or less were found for all six iron-sulfur proteins reflecting internal Trp motion. In addition, some proteins exhibit longer correlation times reflecting segmental motion and overall protein tumbling. The ultrarapid fluorescence decay in iron-sulfur proteins indicates efficient radiationless energy transfer between distant tryptophan residues and iron-sulfur clusters. Such an energy transfer mechanism can be accounted for by referring to the three-dimensional structures of rubredoxin and ferredoxin in calculating the transfer efficiency of the single tryptophan-iron-sulfur couple.

Defects of the respiratory chain in various tissues of old monkeys: a cytochemical-immunocytochemical study.

The aim of the present study was to evaluate if defects of the respiratory chain known to occur in humans, also exist in lower primates. Cytochemical-immunocytochemical studies of the respiratory chain enzymes in five monkeys (10-25 years of age) showed defects of ubiquinone cytochrome-c-oxidoreductase (complex III), of cytochrome-c-oxidase (complex IV) and of ATP-synthase (complex V) in the limb muscles, diaphragm, heart muscle and extraocular muscles of three old animals (about 25 years) and also in the heart muscle of two younger animals (10 and 15 years). Characteristically, the defects were randomly distributed and there was no loss of succinate-dehydrogenase (complex II) in the fibres. Ultracytochemistry-immunocytochemistry of complex IV disclosed that in an involved fibre segment all the mitochondria exhibited the defect. The highest number of defects was observed in the extraocular muscle (up to 340/cm2) while the lowest defect density was present in the limb muscles (2-5/cm2). Defects of complex IV occurred two to three times more often than defects of complex III and besides isolated defects of complex III and IV, combined defects of both complexes were also observed. Defects of complex V occurred exclusively in combination and were rarely seen. Using subunit specific antisera against complex IV, it could be demonstrated at light and electron microscopic level that loss of activity of cytochrome-c-oxidase was associated with a loss both of mitochondrially and nuclearly coded subunits of the enzyme. In summary, aging in lower primates and humans is characterised by a highly similar defect expression of the respiratory chain enzymes, with intercellular and interorgan differences of the aging process, underlining the universal nature of the involved pathogenetic mechanisms.

Defects of the respiratory chain in oxyphil and chief cells of the normal parathyroid and in hyperfunction.

Immunohistochemical detection of complex HIII (ubiquinone- cytochrome-c-oxidoreductase) and complex IV (cytochrome-c-oxidase) of the respiratory chain was performed in parathyroids of 164 humans with normal renal function (group I) and in 55 patients with chronic renal insufficiency (group II) obtained at autopsy. In group I, 33 of the 164 cases showed defects of the respiratory chain (20%). Eighty-five percent of the defects occurred in advanced age (> 50 years). In group II, 39 of 55 cases (70%) had defects, and about 70% of the defects occurred after age 50. In both groups, more than 80% of the defects were localized in oxyphil cell nodules. However, not every oxyphil nodule was involved. In group I, selective defects of complex IV predominated and were found in 47 of 86 defects (55%). Combined defects of complexes III and IV were present in 25 of 86 defects (29%). In contrast, in group II combined defects predominated and were found in 45% (107 of 240 defects), whereas single defects of complex IV existed in 38% (93 of 240 defects). The frequency of selective defects of complex III was about 16% to 17% in both groups. In situ hybridization and PCR studies for the detection of the common deletion (4.977 base pairs) and of various point mutations of mitochondrial of (m)DNA revealed no consistent molecular genetic abnormalities. A point mutation in the tRNALeu(UUR) at nucleotide (nt) 3.260 was found in only one probe. The results show that defects of the respiratory chain occur already in normal parathyroids, most probably during cell aging, especially in oxyphil cells and at a higher rate in hyperfunction. The high predominance of respiratory chain defects in oxyphil cells and their random distribution favors mutations of mtDNA as a possible cause of oxyphilic cell transformation and of the respiratory chain defects. However, the mutations of mtDNA in the parathyroids are apparently different from those in other ageing tissues.