Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor-resistant mutants.

The kinetic and circular dichroic properties of two yeast mutants that are resistant towards specific inhibitors of the mitochondrial cytochrome bc1 complex have been characterized. Both of these mutants have an altered cytochrome b gene in which aromatic residues are exchanged with non-polar residues in a highly conserved region of the protein. The mutant resistant to myxothiazol and mucidin that contains the substitution Phe129----Leu is not greatly affected either in its ubiquinol:cytochrome c reductase or in the spectral properties of cytochrome b. On the other hand, the mutant resistant to stigmatellin that contains the substitution Ile147----Phe shows a large decrease of the catalytic efficiency for ubiquinol and of the maximal turnover of its reductase activity. This stigmatellin mutant also shows an altered circular-dichroic spectrum of the low-potential haem of cytochrome b. This study provides biochemical and biophysical information for identifying a region in mitochondrial cytochrome b that may fulfill a crucial role in the binding of ubiquinol to the bc1 complex. The results are discussed also in terms of the structural model of cytochrome b having a core of four transmembrane helices.     

The structure of the dihaem cytochrome b of fumarate reductase in Wolinella succinogenes: circular dichroism and sequence analysis studies

The fumarate reductase from Wolinella succinogenes contains two haem groups with markedly different midpoint potentials (-20 mV and -200 mV). The enzyme is made up of three subunits, the lipophilic one of which (cytochrome b) ligates the haems. Circular dichroism (CD) spectroscopy has been applied to the reductase in order to obtain information on the structure of the haems and of their environment. This approach is integrated with amino acid sequence comparison of the cytochrome b with other quinone-reacting membrane haemoproteins for predicting the axial ligands of the haems as well as their location relative to the membrane. The following results have been obtained: (1) the CD spectra in the Soret region show exciton coupling indicating haem-haem interaction, which is particularly evident in the reduced state and disappears upon denaturation of the enzyme; (2) The apoprotein of cytochrome b is predicted to consist of five hydrophobic helices (helices A-D and cd), four of which should span the membrane. Helices A, B, C and cd contain a histidine residue each which possibly forms one of the ligands of the haems. It is proposed that haem b (-20 mV) is ligated by H44 and H93, and haem b (-200 mV) by H143 and H182.     

Cloning,heterologous expression,and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris

The cytochrome (cyt) c', cyt c(556), and cyt c(2) genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c' and cyt c(556) have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b(562), in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe(II) and Fe(III) states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe(II)-cyt c(556) is more stable than the corresponding state of Fe(III)-cyt c(556) (DeltaDeltaG(f)(o) =22 kJ/mol).     

Studies on the topology of the protein import channel in relation to the plant mitochondrial processing peptidase integrated into the cytochrome bc1 complex

The mitochondrial processing peptidase (MPP) specifically cleaves N-terminal targeting signals from hundreds of nuclear-encoded, matrix-targeted precursor proteins. In contrast to yeast and mammals, the plant MPP is an integral component of the respiratory cytochrome bc1 complex. The topology of the protein import channel in relation to MPP/bc1 in plants was studied using chimeric precursors containing truncated cytochrome b2 (cyt b2) proteins of 55-167 residues in length, fused to dihydrofolate reductase (DHFR). The DHFR domain could be tightly folded by methotrexate (MTX), generating translocation intermediates trapped in the import channel with only the cyt b2 pre-sequence/mature domain protruding into the matrix. Spinach and soybean mitochondria imported and processed unfolded precursors. MTX-folded intermediates were not processed in spinach but the longest (1-167) MTX-folded cyt b2-DHFR construct was processed in soybean, while yeast mitochondria successfully processed even shorter MTX-folded constructs. The MTX-folded precursors were cleaved with high efficiency by purified spinach MPP/bc1 complex. We interpret these results as indicating that the protein import channel is located distantly from the MPP/bc1 complex in plants, and that there is no link between protein translocation and protein processing.     

Resolution of the circular dichroism spectra of the mitochondrial cytochrome bc1 complex

The circular dichroic spectrum of the mitochondrial cytochrome bc1 complex isolated from bovine heart has been resolved into the contributions from the prosthetic groups: cytochrome c1, the 'Rieske' iron-sulphur centre and the two b cytochromes. It is apparent that firstly, the circular dichroism (CD) properties of cytochrome c1 within the bc1 complex differ from those found in the isolated cytochrome c1 and secondly, both the oxidized and reduced b cytochromes exhibit an intense spectrum of bilobic shape, with the wavelengths of the cross-over points closely corresponding to those of the maxima in the optical absorbance spectra. These latter CD features are discussed in relation to the proposed structure of cytochrome b.     

Mitochondrial Genetics of Chlamydomonas Reinhardtii: Resistance Mutations Marking the Cytochrome B Gene

We describe the genetic and molecular analysis of the first non-Mendelian mutants of Chlamydomonas reinhardtii resistant to myxothiazol, an inhibitor of the respiratory cytochrome bc1 complex. Using a set of seven oligonucleotide probes, restriction fragments containing the mitochondrial cytochrome b (cyt b) gene from C. reinhardtii were isolated from a mitochondrial DNA library. This gene is located adjacent to the gene for subunit 4 of the mitochondrial NADH-dehydrogenase (ND4), near one end of the 15.8-kb linear mitochondrial genome of C. reinhardtii. The algal cytochrome b apoprotein contains 381 amino-acid residues and exhibits a sequence similarity of about 59% with other plant cytochrome b proteins. The cyt b gene from four myxothiazol resistant mutants of C. reinhardtii was amplified for DNA sequence analysis. In comparison to the wild-type strain, all mutants contain an identical point mutation in the cyt b gene, leading to a change of a phenylalanine codon to a leucine codon at amino acid position 129 of the cytochrome b protein. Segregation analysis in tetrads from reciprocal crosses of mutants with wild type shows a strict uniparental inheritance of this mutation from the mating type minus parent (UP-). However, mitochondrial markers from both parents are recovered in vegetative diploids in variable proportions from one experiment to the next for a given cross. On the average, a strong bias is seen for markers inherited from the mating type minus parent.     

The petite phenotype resulting from a truncated copy of subunit 6 results from loss of assembly of the cytochrome bc1 complex and can be suppressed by overexpression of subunit 9.

Disruption of the gene for subunit 6 of the yeast cytochrome bc1 complex (QCR6) causes a temperature-sensitive petite phenotype in contrast to deletion of the coding region of QCR6, which shows no growth defect. Mitochondria from the petite strain carrying the disruption allele were devoid of ubiquinol-cytochrome c oxidoreductase activity but retained cytochrome c oxidase and oligomycin-sensitive ATPase activities. Optical spectra of cytochromes in mitochondrial membranes from the petite strain lacked a cytochrome b absorption band and had a reduced amount of cytochrome c1. Analysis of mitochondrial translation products showed normal synthesis of cytochrome b. Western analysis of mitochondrial membranes from this disruption strain indicates core protein 1 of the cytochrome bc1 complex is present in normal amounts, while cytochrome c1, the Rieske iron-sulfur protein, subunit 6, and subunit 7 were absent or present in very low amounts. Taken together, these findings indicate a loss of assembly of the cytochrome bc1 complex. High copy suppressors of the disruption strain were selected. Two separate families of suppressors were found. The first contained QCR6. The second family consisted of overlapping clones of a second gene distinct from QCR6. These plasmids contained QCR9, the gene which codes for subunit 9 of the yeast cytochrome bc1 complex. Suppression of the QCR6 disruption strain by overexpression of QCR9 indicates a critical interaction between these two proteins in the assembly of the cytochrome bc1 complex.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.     

QO site deficiency can be compensated by extragenic mutations in the hinge region of the iron-sulfur protein in the bc1 complex of Saccharomyces cerevisiae

The mitochondrial bc(1) complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c. The cyt b mutation A144F has been introduced in yeast by the biolistic method. This residue is located in the cyt b cd(1) amphipathic helix in the quinol-oxidizing (Q(O)) site. The resulting mutant was respiration-deficient and was affected in the quinol binding and electron transfer rates at the Q(O) site. An intragenic suppressor mutation was selected (A144F+F179L) that partially alleviated the defect of quinol oxidation of the original mutant A144F. The suppressor mutation F179L, located at less than 4 A from A144F, is likely to compensate directly the steric hindrance caused by phenylalanine at position 144. A second set of suppressor mutations was obtained, which also partially restored the quinol oxidation activity of the bc(1) complex. They were located about 20 A from A144F in the hinge region of the iron-sulfur protein (ISP) between residues 85 and 92. This flexible region is crucial for the movement of the ISP between cyt b and cyt c(1) during enzyme turnover. Our results suggested that the compensatory effect of the mutations in ISP was due to the repositioning of this subunit on cyt b during quinol oxidation. This genetic and biochemical study thus revealed the close interaction between the cyt b cd(1) helix in the quinol-oxidizing Q(O) site and the ISP via the flexible hinge region and that fine-tuning of the Q(O) site catalysis can be achieved by subtle changes in the linker domain of the ISP.     

Effect of famoxadone on photoinduced electron transfer between the iron-sulfur center and cytochrome c1 in the cytochrome bc1 complex

Famoxadone is a new cytochrome bc(1) Q(o) site inhibitor that immobilizes the iron-sulfur protein (ISP) in the b conformation. The effects of famoxadone on electron transfer between the iron-sulfur center (2Fe-2S) and cyt c(1) were studied using a ruthenium dimer to photoinitiate the reaction. The rate constant for electron transfer in the forward direction from 2Fe-2S to cyt c(1) was found to be 16,000 s(-1) in bovine cyt bc(1). Binding famoxadone decreased this rate constant to 1,480 s(-1), consistent with a decrease in mobility of the ISP. Reverse electron transfer from cyt c(1) to 2Fe-2S was found to be biphasic in bovine cyt bc(1) with rate constants of 90,000 and 7,300 s(-1). In the presence of famoxadone, reverse electron transfer was monophasic with a rate constant of 1,420 s(-1). It appears that the rate constants for the release of the oxidized and reduced ISP from the b conformation are the same in the presence of famoxadone. The effects of famoxadone binding on electron transfer were also studied in a series of Rhodobacter sphaeroides cyt bc(1) mutants involving residues at the interface between the Rieske protein and cyt c(1) and/or cyt b.     

Isolation of membrane protein subunits in their native state: evidence for selective binding of chlorophyll and carotenoid to the b(6) subunit of the cytochrome b(6)f complex.

Cytochrome (cyt) b-c complexes play a central role in electron transfer chains and are almost ubiquitous in nature. Although similar in their basic structure and function, the cyt b(6)f complex of photosynthetic membranes and its counterpart, the mitochondrial cyt bc(1) complex, show some characteristic differences which cannot be explained by the high resolution structure of the cyt bc(1) complex alone. Especially the presence of a chlorophyll molecule is a striking feature of all cyt b(6)f complex preparations described so far, imposing questions as to its structural and functional role. To allow a more detailed characterization, we here report the preparation of native subunits cyt b(6) and IV starting from a monomeric cyanobacterial cyt b(6)f complex. Spectroscopical and reversed-phase HPLC analyses of the purified cyt b(6) subunit showed that it contained not only two b-type hemes, but also one chlorophyll a molecule and a cyanobacterial carotenoid, echinenone. Evidence for selective binding of both pigments to this subunit is presented and their putative function is discussed.     

The crystal structure of mitochondrial cytochrome bc1 in complex with famoxadone: the role of aromatic-aromatic interaction in inhibition

Ubiquinol cytochrome c oxido-reductase (EC. 1.10.2.2, bc1) is an integral membrane protein complex essential to cellular respiration. Structures of the 11-subunit mitochondrial bc1 complex were determined with and without the fungicide famoxadone. Specific inhibition by famoxadone is achieved through a coordinated optimization of aromatic-aromatic interactions where conformational rearrangements in famoxadone and in residues lining the inhibitor-binding pocket produce a network of aromatic-aromatic interactions that mimic the crystal lattice of benzene. The profound aromatic-aromatic interactions as supported by prior mutagenesis provide a structural basis for specific protein-ligand interaction in a hydrophobic environment. Dramatic conformational changes, both in cyt. b and ISP subunits in the inhibitor-protein complex, confer experimental evidence for a functional role of cytochrome b in the induced conformational arrest of ISP and allow the identification of a possible intrasubunit signal transduction pathway that controls the movement of ISP. These results support an inhibitory mechanism that is consistent with the requirement for ISP movement in the electron transfer of this complex.     

Net hepatic and splanchnic metabolism of lactate, pyruvate and propionate in dairy cows in vivo in relation to lactation and nutrient supply.

The significance of the exposed haem edge in cytochrome c was directly probed by chemically modifying the partially exposed haem propionate in the crevice region around residues threonine-78 and threonine-49. Reaction of tuna heart cytochrome c with a water-soluble carbodi-imide at pH 3.7 in the absence of any added nucleophilic base leads to the covalent addition of substituted N-acylureas to the protein at two sites. One site has been shown to be a haem propionate by isotope-tracer and i.r.-spectral analysis of haem purified from the apoprotein. The other site is aspartial acid-62 on the back of the molecule. The modified cytochrome c demonstrates abnormal properties, including auto-oxidizability, a reduction potential of + 105mV, a reversible transition to a high-spin species below pH 5.3, no 695 nm charge-transfer band in the ferric state and abnormal binding to mitochondrial membranes. The derivative does react with cytochrome oxidase in deoxycholate-treated submitochondrial particles or in purified preparations with a specific activity of 43-65% compared with that obtained with native cytochrome c. The results are consistent with the view that an intact haem crevice is essential for normal values for physiochemical characteristics, but the significant residual enzymic activity suggests that the electron-transfer interface and/or the cytochrome oxidase-binding site cannot be localized solely in the region of the exposed haem propionate.     

The inhibitor DBMIB provides insight into the functional architecture of the Qo site in the cytochrome b6f complex

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.     

Circular dichroic properties of the tyrosine residues in tetrazole analogues of opioid peptides.

CD studies on tetrazole analogues of opioid peptides show that peptides sharing the same N-terminal sequence, H-TyrPsi[CN(4)]Gly-, give very large Cotton effects of the Tyr side chain in the near-UV region. CD spectra of five such peptides: H-TyrPsi[CN(4)]Gly-Gly-Phe-Leu-OH (I), H-TyrPsi[CN(4)]Gly-Phe-Pro-Gly-Pro-Ile-NH(2) (II), H-TyrPsi[CN(4)]Gly-Phe-Pro-NH(2) (III), H-TyrPsi[CN(4)]Gly-Phe-Gly-Tyr-Pro-Ser-NH(2) (IV), and H-TyrPsi[CN(4)]Gly-Phe-Asp-Val-Val-Gly-NH(2) (V), and two others for comparison: H-Tyr-GlyPsi[CN(4)]Gly-Phe-Leu-OH (VI) and H-TyrPsi[CN(4)]Ala-Phe-Gly-Tyr-Pro-Ser-NH(2) (VII), were measured in methanol, 2,2,2-trifluoroethanol, and water at different pH values. The spectra show that the conformations of the Tyr(1) residue in peptides I-V are very similar in all solvents used but differ distinctly from those observed for VI and VII. Strong Tyr bands in the aromatic region result probably from the rigid structure of the common N-terminal part of peptides I-V. These bands are weaker for IV, which maybe due to the presence of a second Tyr residue in that peptide, giving an opposite contribution to the CD spectrum as that arising from Tyr1. It seems that the rigid structure of the N-terminal part of I-V results from the interaction of the Tyr(1) side chain and the tetrazole ring. The CD bands of the Tyr residues of VI and VII are much smaller than those of I-V in all solvents, except VII in trifluoroethanol (TFE) where Tyr bands comparable in intensity to those of I-V are observed. This spectral property may derive from the same sign contribution of both Tyr residues of VII to the CD spectrum.     

Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments

Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.     

The identification of cation-binding domains on the surface of microsomal cytochrome b5 using 1H-NMR paramagnetic difference spectroscopy.

One-dimensional and two-dimensional 1H-NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethylenediamine) chromium(III) [Cr(en)3(3+)] to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T = 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites II and III are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)3(3+) and cytochrome b5 using two-dimensional double-quantum-filtered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one-dimensional experiments. In a binary complex with ferricytochrome c, Cr(en3(3+) broadens many cytochrome b45 resonances including the haem propionates, His26, Ala54, Thr55 and His80. Although the pattern of line-broadening of resonances at sites II and III is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations.     

Substitution of the sixth axial ligand of Rhodobacter capsulatus cytochrome c1 heme yields novel cytochrome c1 variants with unusual properties.

The cytochrome (cyt) c1 heme of the ubihydroquinone:cytochrome c oxidoreductase (bc1 complex) is covalently attached to two cysteine residues of the cyt c1 polypeptide chain via two thioether bonds, and the fifth and sixth axial ligands of its iron atom are histidine (H) and methionine (M), respectively. The latter residue is M183 in Rhodobacter capsulatus cyt c1, and previous mutagenesis studies revealed its critical role for the physicochemical properties of cyt c1 [Gray, K. A., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873]. In the homologous chloroplast b6f complex, the sixth axial ligand is provided by the amino group of the amino terminal tyrosine residue. To further pursue our investigation on the role played by the sixth axial ligand in heme-protein interactions, novel cyt c1 variants with histidine-lysine (K) and histidine-histidine axial coordination were sought. Using a R. capsulatus genetic system, the cyt c1 mutants M183K and M183H were constructed by site-directed mutagenesis, and chromatophore membranes as well as purified bc1 complexes obtained from these mutants were characterized in detail. The studies revealed that these mutants incorporated the heme group into the mature cyt c1 polypeptides, but yielded nonfunctional bc1 complexes with unusual spectroscopic and thermodynamic properties, including shifted optical absorption maxima (lambdamax) and decreased redox midpoint potential values (Em7). The availability and future detailed studies of these stable cyt c1 mutants should contribute to our understanding of how different factors influence the physicochemical and folding properties of membrane-bound c-type cytochromes in general.     

Spectral analysis of the bc(1) complex components in situ: beyond the traditional difference approach

The cytochrome (cyt) bc(1) complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc(1) complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc(1) complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc(1) turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b(L). From LS analysis of the chromophoric components (RC, c(tot), b(H) and b(L)), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc(1) complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.     

Zinc inhibition of bacterial cytochrome bc(1) reveals the role of cytochrome b E295 in proton release at the Q(o) site

The cytochrome (cyt) bc(1) complex (cyt bc(1)) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc(1) is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn(2+)-induced inhibition of Rhodobacter capsulatus cyt bc(1) using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn(2+) binding mechanism and its inhibitory effect on cyt bc(1) function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn(2+) on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn(2+) binding affinity. The kinetic study showed that wild-type cyt bc(1) and its E295V mutant have similar levels of apparent K(m) values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K(I) values for Zn(2+) are 8.3 and 38.5 μM, respectively. The calorimetry-based K(D) values for the high-affinity site of cyt bc(1) are on the same order of magnitude as the K(I) values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn(2+), whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn(2+). Our overall results indicate that the proton-active E295 residue near the Q(o) site of cyt bc(1) can bind directly to Zn(2+), resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Q(o) site of cyt bc(1).