Biomedical subjects

Artur Osyczka

Publications and source records attributed to Artur Osyczka.

12 recordsLinked to original sources

PubMed2006

Resilience of Rhodobacter sphaeroides cytochrome bc1 to heme c1 ligation changes.

Typically, c hemes are bound to the protein through two thioether bonds to cysteines and two axial ligands to the heme iron. In high-potential class I c-type cytochromes, these axial ligands are commonly His-Met. A change in this methionine axial ligand is often correlated with a dramatic drop in the heme redox potential and loss of function. Here we describe a bacterial cytochrome c with an unusual tolerance to the alternations in the heme ligation pattern. Substitution of the heme ligating methionine (M185) in cytochrome c1 of the Rhodobacter sphaeroides cytochrome bc1 complex with Lys and Leu lowers the redox midpoint potential but not enough to prevent physiologically competent electron transfer in these fully functional variants. Only when Met-185 is replaced with His is the drop in the redox potential sufficiently large to cause cytochrome bc1 electron transfer chain failure. Functional mutants preserve the structural integrity of the heme crevice: only the nonfunctional His variant allows carbon monoxide to bind to reduced heme, indicating a significant opening of the heme environment. This range of cytochrome c1 ligand mutants exposes both the relative resilience to sixth axial ligand change and the ultimate thermodynamic limits of operation of the cofactor chains in cytochrome bc1.

Electron Transport Complex III↗

PubMed2006

Role of the PEWY glutamate in hydroquinone-quinone oxidation-reduction catalysis in the Qo Site of cytochrome bc1.

The glutamic acid residue of the conserved PEWY motif of the Q(o) site of cytochrome bc(1) is widely discussed as central to reversible Q(o) site catalysis of two-electron, two-proton hydroquinone-quinone oxidation-reduction. Extensive mutation of this glutamate (E295) to A, V, F, H, K, and Q in purple photosynthetic Rhodobacter capsulatus results in hydroquinone oxidation rates that are between 5 and 50-fold slower than that in the wild type. However, the mutants show little or no detectable effects on hydroquinone or quinone exchange and binding at the Q(o) site nor on subsequent Q(o) site-mediated redox equilibria in the c-chain and b-chain from pH 5-10. Lack of effects of mutations on the E(m)/pH plots rules out involvement of E295 in the strong electron-proton coupling evident in either the FeS center or heme b(L). These detailed equilibrium and kinetic analyses demonstrate that E295 is not irreplaceable in the Q(o) site catalytic mechanism. Rather, E295 and several other Q(o) site residues that can also be widely varied and still support hydroquinone oxidation illustrate the considerable resilience of Q(o) site activity to mutational change in Q(o) site environs. Residues and water molecules appear to cooperate in providing a physical and chemical environment supporting hydroquinone oxidation rates comparable to those seen in nonprotein aqueous environments at electrodes. We suggest that residues at the Q(o) site (and, possibly, other respiratory and photosynthetic quinone and oxygen binding sites) are a product of natural selection primarily acting not to lower catalytic barriers according to the traditional view of enzymatic catalysis but rather to develop specificity by raising barriers in defense of semiquinone loss or energy wasting short-circuit reactions.

Binding Sites↗

PubMed2006

A functional hybrid between the cytochrome bc1 complex and its physiological membrane-anchored electron acceptor cytochrome cy in Rhodobacter capsulatus.

The membrane integral ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductase (or the cyt bc1 complex) and its physiological electron acceptor, the membrane-anchored cytochrome cy (cyt cy), are discrete components of photosynthetic and respiratory electron transport chains of purple non-sulfur, facultative phototrophic bacteria of Rhodobacter species. In Rhodobacter capsulatus, it has been observed previously that, depending on the growth condition, absence of the cyt bc1 complex is often correlated with a similar lack of cyt cy (Jenney, F. E., et al. (1994) Biochemistry 33, 2496-2502), as if these two membrane integral components form a non-transient larger structure. To probe whether such a structural super complex can exist in photosynthetic or respiratory membranes, we attempted to genetically fuse cyt cy to the cyt bc1 complex. Here, we report successful production, and initial characterization, of a functional cyt bc1-cy fusion complex that supports photosynthetic growth of an appropriate R. capsulatus mutant strain. The three-subunit cyt bc1-cy fusion complex has an unprecedented bis-heme cyt c1-cy subunit instead of the native mono-heme cyt c1, is efficiently matured and assembled, and can sustain cyclic electron transfer in situ. The remarkable ability of R. capsulatus cells to produce a cyt bc1-cy fusion complex supports the notion that structural super complexes between photosynthetic or respiratory components occur to ensure efficient cellular energy production.

Cell Membrane↗

PubMed2005

De novo design of a redox-active minimal rubredoxin mimic.

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins and the de novo design of helical bundles that bind metal ion cofactors. However, the design of metalloproteins containing beta-structure is less well developed, despite the frequent occurrence of beta-conformations in natural metalloproteins. Here, we describe the design and construction of a beta-protein, RM1, that forms a stable, redox-active 4-Cys thiolate Fe(II/III) site analogous to the active site of rubredoxin. The protein folds into a beta-structure in the presence and absence of metal ions and binds Fe(II/III) to form a redox-active site that is stable to repeated cycles of oxidation and reduction, even in an aerobic environment.

Binding Sites↗

PubMed2005

Direct observation of redox-linked histidine protonation changes in the iron-sulfur protein of the cytochrome bc1 complex by ATR-FTIR spectroscopy.

The redox-linked protonation chemistry of the iron-sulfur protein (ISP) of the cytochrome bc(1) complex was studied by analysis of the pH dependencies of redox difference spectra using perfusion/electrochemically induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The ISP of Rhodobacter capsulatus within the intact cytochrome bc(1) complex was analyzed in a mutant form in which the midpoint potential of cytochrome c(1) was lower than that of the ISP. This was compared to a soluble domain of the ISP from the bovine bc(1) complex. Spectra of in situ bacterial and isolated bovine proteins differ markedly only in part of their amide I regions with the in situ protein having additional pH-dependent component(s). Apart from this, both in situ and isolated proteins exhibited the same pH-dependent IR features in reduced minus oxidized difference spectra. Specifically, at high pH, a strong H/D insensitive negative band appeared at 1447/1450 cm(-)(1), together with a peak at 1310 cm(-)(1), the change of a 1267/1255 cm(-)(1) peak/trough into a simple 1266 cm(-)(1) peak, and a trough at 1107 cm(-)(1). Comparison with spectra of model materials indicates that all four signals arise from an imidazolate to imidazole transition of histidine, hence providing the first direct demonstration that histidine is the redox-linked protonation site of the ISP. The 1450 cm(-)(1) band can be assigned to a ring stretch that is unique to the imidazolate form of histidine. It is relatively sharp, has a high extinction coefficient, and provides a novel marker band for the detection of imidazolate intermediates in enzymatic mechanisms generally.

Amino Acid Substitution↗

PubMed2005

Fixing the Q cycle.

Mitchell's key insight that all bioenergetic membranes run on the conversion of redox energy into transmembrane electrical and proton gradients took the form 30 years ago of a working model of the Q cycle of cytochrome bc1, which operates reversibly on coupled electron and proton transfers of quinone at two binding sites on opposite membrane faces. His remarkable model still stands today, but he had no structural information to provide understanding into how dangerous short-circuit redox reactions were avoided. Now, it is clear that the Q cycle must be fixed with a special mechanism that avoids semiquinone-mediated short circuits. Either the redox states of the quinone electron-transfer partners double-gate the semiquinone-intermediate stability, or semiquinone is avoided altogether in concerted double-electron transfer.

Electron Transport↗

PubMed2004

ATR-FTIR spectroscopy studies of iron-sulfur protein and cytochrome c1 in the Rhodobacter capsulatus cytochrome bc1 complex.

Redox transitions in the Rhodobacter capsulatus cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with synchronous visible spectroscopy, in both the wild type and a cytochrome c(1) point mutant, M183K, in which the midpoint potential of heme was lowered from the wild-type value of 320 mV to 60 mV. Overall redox difference spectra of the wild type and M183K mutant were essentially identical, indicating that the mutation did not cause any major structural perturbation. Spectra were compared with data on the bovine bc(1) complex, and tentative assignments of several bands could be made by comparison with available data on model compounds and crystallographic structures. The bacterial spectra showed contributions from ubiquinone that were much larger than in the bovine enzyme, arising from additional bound and adventitious ubiquinone. The M183K mutant enabled selective reduction of the iron-sulfur protein which in turn allowed the IR redox difference spectra of ISP and cytochrome c(1) to be deconvoluted at high signal/noise ratios, and features of these spectra are interpreted in light of structural and mechanistic information.

Cytochromes c1↗

PubMed2004

De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1.

An idealized, water-soluble D(2)-symmetric diheme protein is constructed based on a mathematical parametrization of the backbone coordinates of the transmembrane diheme four-helix bundle in cytochrome bc(1). Each heme is coordinated by two His residues from diagonally apposed helices. In the model, the imidazole rings of the His ligands are held in a somewhat unusual perpendicular orientation as found in cytochrome bc(1), which is maintained by a second-shell hydrogen bond to a Thr side chain on a neighboring helix. The resulting peptide is unfolded in the apo state but assembles cooperatively upon binding to heme into a well-folded tetramer. Each tetramer binds two hemes with high affinity at low micromolar concentrations. The equilibrium reduction midpoint potential varies between -76 mV and -124 mV vs SHE in the reducing and oxidizing direction, respectively. The EPR spectrum of the ferric complex indicates the presence of a low-spin species, with a g(max) value of 3.35 comparable to those obtained for hemes b of cytochrome bc(1) (3.79 and 3.44). This provides strong support for the designed perpendicular orientation of the imidazole ligands. Moreover, NMR spectra show that the protein exists in solution in a unique conformation and is amenable to structural studies. This protein may provide a useful scaffold for determining how second-shell ligands affect the redox potential of the heme cofactor.

Binding Sites↗

PubMed2004

Novel cyanide inhibition at cytochrome c1 of Rhodobacter capsulatus cytochrome bc1.

Oxidized cytochrome c(1) in photosynthetic bacterium Rhodobacter capsulatus cytochrome bc(1) reversibly binds cyanide with surprisingly high, micromolar affinity. The binding dramatically lowers the redox midpoint potential of heme c(1) and inhibits steady-state turnover activity of the enzyme. As cytochrome c(1), an auxiliary redox center of the high-potential chain of cytochrome bc(1), does not interact directly with the catalytic quinone/quinol binding sites Q(o) and Q(i), cyanide introduces a novel, Q-site independent locus of inhibition. This is the first report of a reversible inhibitor that manipulates the energetics and electron transfers of the high-potential redox chain of cytochrome bc(1), while maintaining quinone substrate catalytic sites in an intact form.

Binding Sites↗

PubMed2004

Reversible redox energy coupling in electron transfer chains.

Reversibility is a common theme in respiratory and photosynthetic systems that couple electron transfer with a transmembrane proton gradient driving ATP production. This includes the intensely studied cytochrome bc1, which catalyses electron transfer between quinone and cytochrome c. To understand how efficient reversible energy coupling works, here we have progressively inactivated individual cofactors comprising cytochrome bc1. We have resolved millisecond reversibility in all electron-tunnelling steps and coupled proton exchanges, including charge-separating hydroquinone-quinone catalysis at the Q(o) site, which shows that redox equilibria are relevant on a catalytic timescale. Such rapid reversibility renders popular models based on a semiquinone in Q(o) site catalysis prone to short-circuit failure. Two mechanisms allow reversible function and safely relegate short-circuits to long-distance electron tunnelling on a timescale of seconds: conformational gating of semiquinone for both forward and reverse electron transfer, or concerted two-electron quinone redox chemistry that avoids the semiquinone intermediate altogether.

Adenosine Triphosphate↗

PubMed2003

Role of acidic and aromatic amino acids in Rhodobacter capsulatus cytochrome c1. A site-directed mutagenesis study.

The roles of two evolutionarily conserved aromatic residues in the cytochrome c(1) component of the Rhodobacter capsulatus cytochrome bc(1) complex, phenylalanine 138 and tyrosine 194, were analyzed by site-directed mutagenesis, in combination with biophysical and biochemical measurements. Changing Phe138 to either alanine or valine, but not to tyrosine, results in redox heterogeneity of cytochrome c(1). Replacement of Phe138 by an aliphatic amino acid also caused changes in the EPR spectrum of the cytochrome and resulted in decreases in the steady-state V(max) for the hydroquinone/cytochrome c oxidoreductase activity of cytochrome bc(1) complexes containing the mutated cytochrome c(1). These findings indicate that the presence of an aromatic residue at position 138 is essential for maintaining the native environment of the cytochrome c(1) heme. In contrast, replacement of Tyr194 by aliphatic amino acids had no significant effect on either the E(m) of cytochrome c(1) or the steady-state activity parameters. Site-directed mutagenesis of glutamate and aspartate residues in a conserved acidic patch (region 2) on Rb. capsulatus cytochrome c(1) suggests that these negatively charged residues do not play a role in the docking of cytochrome c(2) with the cytochrome bc(1) complex.

Amino Acids↗

PubMed2002

Spectroscopic and oxidation-reduction properties of Rhodobacter capsulatus cytochrome c1 and its M183K and M183H variants.

Two variants of the cytochrome c1 component of the Rhodobacter capsulatus cytochrome bc1 complex, in which Met183 (an axial heme ligand) was replaced by lysine (M183K) or histidine (M183H), have been analyzed. Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra of the intact complex indicate that the histidine/methionine heme ligation of the wild-type cytochrome is replaced by histidine/lysine ligation in M183K and histidine/histidine ligation in M183H. Variable amounts of histidine/histidine axial heme ligation were also detected in purified wild-type cytochrome c1 and its M183K variant, suggesting that a histidine outside the CSACH heme-binding domain can be recruited as an alternative ligand. Oxidation-reduction titrations of the heme in purified cytochrome c1 revealed multiple redox forms. Titrations of the purified cytochrome carried out in the oxidative or reductive direction differ. In contrast, titrations of cytochrome c1 in the intact bc1 complex and in a subcomplex missing the Rieske iron-sulfur protein were fully reversible. An Em7 value of -330 mV was measured for the single disulfide bond in cytochrome c1. The origins of heme redox heterogeneity, and of the differences between reductive and oxidative heme titrations, are discussed in terms of conformational changes and the role of the disulfide in maintaining the native structure of cytochrome c1.

Amino Acid Substitution↗