Structure-function studies of [2Fe-2S] ferredoxins

The ability to overexpress [2Fe-2S] ferredoxins inEscherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegetative and heterocyst forms ofAnabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in these proteins in their oxidized and reduced states has been studied by¹H,²H,¹³C, and¹⁵N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partnerAnabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).     

Atomic resolution structures of oxidized [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus in two crystal forms: systematic distortion of [4Fe-4S] cluster in the protein

Diffraction data of two crystal forms (forms I and II) of [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus have been collected to 0.92 A and 1.00 A resolutions, respectively, at 100 K using synchrotron radiation. Anisotropic temperature factors were introduced for all non-hydrogen atoms in the refinement with SHELX-97, in which stereochemical restraints were applied to the protein chain but not to the [4Fe-4S] cluster. The final crystallographic R-factors are 9.8 % for 7.0-0.92 A resolution data of the form I and 11.2 % for the 13.3-1.0 A resolution data of the form II. Many hydrogen atoms as well as multiple conformations for several side-chains have been identified. The present refinement has revised the conformations of several peptide bonds and side-chains assigned previously at 2.3 A resolution; the largest correction was that the main-chain of Pro1 and the side-chain of Lys2 were changed by rotating the C(alpha)-C bond of Lys2. Although the overall structures in the two crystal forms are very similar, conformational differences are observed in the two residues at the middle (Glu29 and Asp30) and the C-terminal residues, which have large temperature factors. The [4Fe-4S] cluster is a distorted cube with non-planar rhombic faces. Slight but significant compression of the four Fe-S bonds along one direction is observed in both crystal forms, and results in the D(2d) symmetry of the cluster. The compressed direction of the cluster relative to the protein is conserved in the two crystal forms and consistent with that in one of the clusters in Clostridium acidurici ferredoxin.     

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F_X, F_A and F_B, the last two are bound by the PsaC subunit. We have modelled the F_A and F_B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F_A and F_B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F_A-maquette is − 0.44 ± 0.03 V and in the F_B-maquette is − 0.47 ± 0.03 V. Both are close to that of F_A and F_B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F_A and F_B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]^1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]^1+,2+ clusters termed F_A and F_B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F_B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]^1+,2+ clusters, despite the absence of one of the biological ligands. ¹⁹F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F_B cluster. The finding that site-modified [4Fe-4S]^1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P₂³⁸⁰) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.     

Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc 1 complex inSaccharomyces cerevisiae

The iron-sulfur protein of the cytochromebc ₁ complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochromec ₁ and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochromeb-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochromebc ₁ complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochromec ₁ and generates a strongly reducing ubisemiquinone anion radical which is proximal to theb-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.     

Possible direct involvement of the active-site [4Fe-4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

The GcpE enzyme converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the DOXP pathway for isoprene biosynthesis. Purification of the enzyme under exclusion of air leads to a preparation that contains solely [4Fe-4S] clusters. Kinetic studies showed that in the presence of the artificial reductant dithionite and MEcPP a new transient iron-sulfur-based signal is detected in electron paramagnetic resonance (EPR) spectroscopy. Similarity of this EPR signal to that detected in ferredoxin:thioredoxin reductase indicates that during the reaction an intermediate is directly bound to the active-site cluster.     

Role of cell signaling in B[a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[a]P metabolites

Here we show that several cell signaling inhibitors have effect on cyp1a1 expression and the metabolism of benzo[a]pyrene (B[a]P) in Hepa1c1c7 cells. The CYP1A1 inhibitor alpha-naphthoflavone (alpha-NF), the p53 inhibitor pifithrin-alpha (PFT-alpha), the ERK inhibitors PD98059 and U0126, and the p38 MAPK inhibitors SB202190 and PD169316 induced the expression and level of cyp1a1 protein. On the other hand, during the first h the inhibitors appeared to reduce the metabolism of B[a]P as measured by the generation of tetrols and by covalent binding of B[a]P to macromolecules. In contrast, the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, had neither an effect on the cyp1a1 expression nor the B[a]P-metabolism. In order to avoid these unspecific effects, we characterized the mechanisms involved in the apoptotic effects of B[a]P-metabolites. B[a]P and the B[a]P-metabolites B[a]P-7,8-DHD and BPDE-I induced apoptosis, whereas B[a]P-4,5-DHD had no effect. B[a]P, B[a]P-7,8-DHD and BPDE-I induced an accumulation and phosphorylation of p53, while the Bcl-2 proteins Bcl-xl, Bad and Bid were down-regulated. Interestingly, the levels of anti-apoptotic phospho-Bad were up-regulated in response to B[a]P as well as to B[a]P-7,8-DHD and BPDE-I. Both p38 MAPK and JNK were activated, but the p38 MAPK inhibitors were not able to inhibit BPDE-I-induced apoptosis. PFT-alpha reduced the BPDE-I-induced apoptosis, while both the PI-3 kinase inhibitor and the ERK inhibitors increased the apoptosis in combination with BPDE-I. BPDE-I also triggered apoptosis in primary cultures of rat lung cells. In conclusion, often used cell signaling inhibitors both enhanced the expression and the level of cyp1a1 and more directly acted as inhibitors of cyp1a1 metabolism of B[a]P. However, studies with the B[a]P-metabolite BPDE-I supported the previous suggestion that p53 has a role in the pro-apoptotic signaling pathway induced by B[a]P. Furthermore, these studies also show that the reactive metabolites of B[a]P induce the anti-apoptotic signals, Akt and ERK. Neither the induction nor the activity of p38 MAPK and JNK seems to be of major importance for the B[a]P-induced apoptosis.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

Molecular docking studies of 1-(substituted phenyl)-3-(naphtha [1, 2-d] thiazol-2-yl) urea/thiourea derivatives with human adenosine A(2A) receptor

Computational assessment of the binding interactions of drugs is an important component of computer-aided drug design paradigms. In this perspective, a set of 30 1-(substituted phenyl)-3-(naphtha[1, 2-d] thiazol-2-yl) urea/thiourea derivatives showing antiparkinsonian activity were docked into inhibitor binding cavity of human adenosine A(2A) receptor (AA2AR) to understand their mode of binding interactions in silico. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results signify that the molecular docking approach is reliable and produces a good correlation coefficient (r(2) = 0.483) between docking score and antiparkinsonian activity (in terms of % reduction in catalepsy score). Potent antiparkinsonian agents carried methoxy group in the phenyl ring, exhibited both hydrophilic and lipophilic interactions with lower energy of binding at the AA(2A)R. These molecular docking analyses should, in our view, contribute for further development of selective AA(2A)R antagonists for the treatment of Parkinson's disease.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

Structure and Function of the Bacterial bc 1 Complex: Domain Movement, Subunit Interactions, and Emerging Rationale Engineering Attempts

The ubiquinol: cytochrome c oxidoreductase, or the bc ₁ complex, is a key component ofboth respiratory and photosynthetic electron transfer and contributes to the formation of anelectrochemical gradient necessary for ATP synthesis. Numerous bacteria harbor a bc ₁ complexcomprised of three redox-active subunits, which bear two b-type hemes, one c-type heme, andone [2Fe–2S] cluster as prosthetic groups. Photosynthetic bacteria like Rhodobacter speciesprovide powerful models for studying the function and structure of this enzyme and are beingwidely used. In recent years, extensive use of spontaneous and site-directed mutants and theirrevertants, new inhibitors, discovery of natural variants of this enzyme in various species, andengineering of novel bc ₁ complexes in species amenable to genetic manipulations have providedus with a wealth of information on the mechanism of function, nature of subunit interactions,and assembly of this important enzyme. The recent resolution of the structure of variousmitochondrial bc ₁ complexes in different crystallographic forms has consolidated previousfindings, added atomic-scale precision to our knowledge, and raised new issues, such as thepossible movement of the Rieske Fe–S protein subunit during Q_o site catalysis. Here, studiesperformed during the last few years using bacterial bc ₁ complexes are reviewed briefly andongoing investigations and future challenges of this exciting field are mentioned.     

The [FeFe]-hydrogenase maturase HydF from Clostridium acetobutylicum contains a CO and CN ligated iron cofactor

Biosynthesis of the [FeFe] hydrogenases active site (H-cluster) requires three maturation factors whose respective roles are not understood yet. The clostridial maturation enzymes (CaHydE, CaHydF and CaHydG) were homologously overexpressed in their native host Clostridium acetobutylicum. CaHydF was able to activate Chlamydomonas reinhardtii [FeFe] hydrogenase apoprotein (CrHydA1_apo) to almost 100% compared to the native specific hydrogen evolution activity. Based on electron paramagnetic resonance spectroscopy and Fourier-transform infrared spectroscopy data the existence of a [4Fe4S] cluster and a CO and CN⁻ ligand coordinated di-iron cluster is suggested. This study contains the first experimental evidence that the bi-nuclear part of the H-cluster is assembled in HydF.     

The role of repair protein Rad51 in synergistic cytotoxicity and mutagenicity induced by epidermal growth factor receptor inhibitor (Gefitinib, IressaR) and benzo[a]pyrene in human lung cancer

Rad51 protein is essential for homologous recombination repair of DNA damage, and is over-expressed in chemo- or radioresistant carcinomas. The polycyclic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) affects MAPKs transduction pathways. Gefitinib (IressaR, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and ERK1/2 activation. We hypothesized that gefitinib enhances B[a]P-mediated cytotoxicity by decreasing ERK1/2 activation. Exposure of human lung cancer cells to gefitinib decreased B[a]P-elicited ERK1/2 activation and induced Rad51 protein expression. Gefitinib and B[a]P co-treatment decreased Rad51 protein stability by triggering degradation via a 26S proteasome-dependent pathway. Expression of constitutive active MKK1/2 vectors (MKK1/2-CA) rescues the decreased ERK1/2 activity, and restores Rad51 protein level and stability under gefitinib and B[a]P co-treatment. Gefitinib enhances B[a]P-induced growth inhibition, cytotoxicity and mutagenicity. Co-treatment with gefitinib and B[a]P can further inhibit cell growth significantly after depletion of endogenous Rad51 by siRad51 RNA transfection. Enhancement of ERK1/2 activation by MKK1-CA expression decrease B[a]P- and gefitinib-induced cytotoxicity, and B[a]P-induced mutagenicity. Rad51 protein protects lung cancer cells from synergistic cytotoxic and mutagenic effects induced by gefitinib and B[a]P. Suppression of Rad51 protein expression may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib.     

Co-exposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX

Phosphorylation of histone H2AX (termed gamma-H2AX) was recently identified as an early event after induction of DNA double strand breaks (DSBs). We have previously shown that co-exposure to benzo[a]pyrene (BaP), a wide-spread environmental carcinogen, and ultraviolet A (UVA), a major component of solar UV radiation, induced DSBs in mammalian cells. In the present study, we examined whether co-exposure to BaP and UVA generates gamma-H2AX in CHO-K1 cells. Single treatment with BaP (10(-9)-10(-7)M) or UVA ( approximately 2.4 J/cm(2)) did not result in gamma-H2AX, however, co-exposure drastically induced foci of gamma-H2AX in a dose-dependent manner. gamma-H2AX could be detected even at very low concentration of BaP (10(-9)M) plus UVA (0.6J/cm(2)), which did not change cell survival rates. NaN(3) effectively inhibited the formation of gamma-H2AX induced by co-exposure, indicating the contribution of singlet oxygen. This is the first evidence that co-exposure to BaP and UVA induced DSBs, involving gamma-H2AX.     

Potentiometric and spectroscopic studies on the solution molecular structures of copper(II)-[S]-2-[N-(2′-hydroxybenzyl)aminomethyl]pyrrolidine complexes, potential non-steroidal anti-inflammatory agents (NSAIDs)

The purpose of this study was to investigate the complexes formed by copper(II) with potential non-steroidal anti-inflammatory agents (NSAIDs) under physiological conditions. A former study suggested that 2-benzylaminomethylpyrrolidine ligands could be good candidates as potential OIL (OH-inactivating ligand) when complexed to copper(II). In order to assess the chemical behavior as OIL, [S]-2-[N-(2′-hydroxybenzyl)aminomethyl]pyrrolidine (OHbamp) was synthesized and bound to copper(II). Physico-chemical properties were determined at 37 °C in 0.15 M NaCl using glass electrode potentiometry, UV-Vis and circular dichroism spectroscopies, before and after copper(II) complexation. [Cu(OHbamp)(H₂O)₃]⁺ was the main complex found at both physiological and inflammatory pH values, showing appreciable stability at pathological pH compared to copper(II) complexes of histidine, the predominant low-molar-mass ligand of copper(II) in blood plasma. However, neutral species such as [Cu(OHbamp)₂(H₂O)₂] and [Cu(OHbamp)(OH)(H₂O)₃] are predominant only above pH 8, preventing a significant amount of drug from diffusing through membranes at inflammatory pH. In conclusion, copper(II)-OHbamp system does not meet all the requirements to be an OIL. Nevertheless, these results allow us to better identify the chemical features needed for a good OIL candidate.     

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H₂O₂, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 °C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2 × 10⁴ M^− 1 s^− 1, 30 M^− 1 s^− 1, 3.4 × 10³ M^− 1 s^− 1, and 8.6 × 10³ M^− 1 s^− 1, respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.     

Spectral analysis of the bc1 complex components in situ: Beyond the traditional difference approach

The cytochrome (cyt) bc₁ 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 bc₁ 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 bc₁ 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 bc₁ 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 bc₁ complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.