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1.
Science ; 366(6461): 100-104, 2019 10 04.
Article in English | MEDLINE | ID: mdl-31604309

ABSTRACT

Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the Escherichia coli cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site.


Subject(s)
Cytochrome b Group/chemistry , Electron Transport Chain Complex Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Oxidoreductases/chemistry , Catalytic Domain , Cryoelectron Microscopy , Heme/chemistry , Models, Molecular , Oxidation-Reduction , Oxygen/chemistry , Protein Structure, Quaternary , Protein Subunits/chemistry , Protons , Ubiquinone/chemistry
2.
J Inorg Biochem ; 108: 196-202, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22197476

ABSTRACT

This study compares the behavior as cytochrome c oxidase (CcO) functional and structural models of a series of reported and unreported ligands that provide either a binding site for copper without a built-in proximal base, or both a flexible binding site for copper and a built-in proximal base, or a fixed binding site for copper with a built-in proximal base. The comparisons of the models show that the relative position of the two metal sites is not only a crucial parameter in the control of the catalytic behavior but also essential in mimicking other features of the enzyme such as CO exchange between the ferrous heme a(3) and the cuprous Cu(B) center.


Subject(s)
Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Binding Sites , Copper/chemistry , Electrochemistry/methods , Heme/chemistry , Metals/chemistry
3.
Biochem Soc Trans ; 33(Pt 4): 836-9, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16042610

ABSTRACT

The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of ions across the membrane. It was assumed that the complex exclusively works as a proton pump. Recently, it has been proposed that complex I from Klebsiella pneumoniae and Escherichia coli work as Na+ pumps. We have used an E. coli complex I preparation to determine the type of ion(s) translocated by means of enzyme activity, generation of a membrane potential and redox-induced Fourier-transform infrared spectroscopy. We did not find any indications for Na+ translocation by the E. coli complex I.


Subject(s)
Electron Transport Complex I/metabolism , Escherichia coli/enzymology , Biological Transport , Electron Transport Complex I/antagonists & inhibitors , Escherichia coli Proteins/metabolism , Ions/metabolism , Kinetics , Spectroscopy, Fourier Transform Infrared
4.
Biopolymers ; 74(1-2): 73-6, 2004.
Article in English | MEDLINE | ID: mdl-15137098

ABSTRACT

In this study we present the electrochemically induced Fourier transform infrared (FTIR) difference spectra of the Cu(A) center derived from the ba(3)-type cytochrome c oxidase of Thermus thermophilus in the spectral range from 1800 to 500 cm(-1). The mid infrared is dominated by the nu(C[double bond]O) vibrations of the amide I modes at 1688, 1660, and 1635 cm(-1), reflecting the redox-induced perturbation of the predominantly beta-sheet type structure. The corresponding amide II signal is found at 1528 cm(-1). In the lower frequency range below 800 cm(-1), modes from amino acids liganding the Cu(A) center are expected. On the basis of the absorbance spectrum of the isolated amino acids, methionine is identified as an important residue, displaying C-S vibrations at these frequencies. This spectral range was previously disregarded by protein IR spectroscopists, mainly due to the strong absorbance of the solvent, H(2)O. With an optimized setup, however, IR is found suitable for structure/function studies on proteins.


Subject(s)
Electron Transport Complex IV/chemistry , Spectrophotometry/methods , Spectroscopy, Fourier Transform Infrared/methods , Thermus thermophilus/enzymology , Amino Acids/chemistry , Electrochemistry , Oxidation-Reduction , Spectrophotometry, Infrared , Water/chemistry
5.
Biochemistry ; 40(29): 8548-56, 2001 Jul 24.
Article in English | MEDLINE | ID: mdl-11456494

ABSTRACT

Cytochrome bd is one of the two quinol oxidases in the respiratory chain of Escherichia coli. The enzyme contains three heme prosthetic groups. The dioxygen binding site is heme d, which is thought to be part of the heme-heme binuclear center along with heme b(595), which is a high-spin heme whose function is not known. Protein sequence alignments [Osborne, J. P., and Gennis, R. B. (1999) Biochim. Biophys Acta 1410, 32--50] of cytochrome bd quinol oxidase sequences from different microorganisms have revealed a highly conserved sequence (GWXXXEXGRQPW; bold letters indicate strictly conserved residues) predicted to be on the periplasmic side of the membrane between transmembrane helices 8 and 9 in subunit I. The functional importance of this region is investigated in the current work by site-directed mutagenesis. Several mutations in this region (W441A, E445A/Q, R448A, Q449A, and W451A) resulted in a catalytically inactive enzyme with abnormal UV--vis spectra. E445A was selected for detailed analysis because of the absence of the absorption bands from heme b(595). Detailed spectroscopic and chemical analyses, indeed, show that one of the three heme prosthetic groups in the enzyme, heme b(595), is specifically perturbed and mostly missing from this mutant. Surprisingly, heme d, while known to interact with heme b(595), appears relatively unperturbed, whereas the low-spin heme b(558) shows some modification. This is the first report of a mutation that specifically affects the binding site of heme b(595).


Subject(s)
Cytochromes/genetics , Electron Transport Chain Complex Proteins , Escherichia coli Proteins , Escherichia coli/enzymology , Heme/analogs & derivatives , Heme/chemistry , Mutagenesis, Site-Directed , Oxidoreductases/genetics , Alanine/genetics , Amino Acid Sequence , Carbon Monoxide/chemistry , Conserved Sequence/genetics , Cyanides/chemistry , Cytochrome b Group , Cytochromes/chemistry , Electrochemistry , Electron Spin Resonance Spectroscopy , Escherichia coli/genetics , Glutamic Acid/genetics , Molecular Sequence Data , Oxidation-Reduction , Oxidoreductases/chemistry , Oxidoreductases, N-Demethylating/chemistry , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Quinone Reductases/chemistry , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman
6.
Spectrochim Acta A Mol Biomol Spectrosc ; 57A(5): 1123-31, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11374571

ABSTRACT

In this work we present the separation of FTIR difference signals induced by electron transfer to/from the redox centers of the cytochrome c oxidase from P. denitrificans and compare electrochemically induced FTIR difference spectra with those induced by CO photolysis. FTIR difference spectra of rebinding of CO to the half reduced (mixed valence) form of the cytochrome c oxidase after photolysis reflect the conformational changes induced by the rebinding of CO and by electron transfer reactions from heme a3 to heme a and further on to CUA. During this process, heme a3 (and CUB) are oxidized, whereas heme a and CuA are reduced. By subtracting these difference spectra from an electrochemically induced FTIR difference spectrum, where all four cofactors are reduced, the contributions for heme a3 (and CuB) could be separated. Correspondingly, the spectral contributions of heme a and CuA have been separated. The comparison of these spectra with the spectra calculated for the hemes on the basis of their redox dependent changes previously published in Hellwig et al., (Biochemistry 38, (1999) 1685-1694) show a high degree of similarity, except for additional signals coupled to the reorganization of the binuclear center upon CO rebinding. The separated spectra clearly show that the signals attributed to Glu278, an amino acid discussed to be crucial for proton pumping, is coupled to electron transfer to/from heme a and the binuclear heme a3-CuB center.


Subject(s)
Electron Transport Complex IV/chemistry , Glutamic Acid/chemistry , Heme/chemistry , Paracoccus denitrificans/enzymology , Electron Transport , Heme/analogs & derivatives , Oxidation-Reduction , Protein Conformation , Spectroscopy, Fourier Transform Infrared
7.
Biochemistry ; 40(4): 1077-82, 2001 Jan 30.
Article in English | MEDLINE | ID: mdl-11170431

ABSTRACT

Aspartate-75 (D75) was recently suggested to participate in a ubiquinone-binding site in subunit I of cytochrome bo(3) from Escherichia coli on the basis of a structural model [Abramson, J., Riistama, S., Larsson, G., Jasaitis, A., Svensson-Ek, M., Laakkonen, L., Puustinen, A., Iwata, S., and Wikström, M. (2000) Nat. Struct. Biol. 7 (10), 910-917]. We studied the protonation state of D75 for the reduced and oxidized forms of the enzyme, using a combined site-directed mutagenesis, electrochemical, and FTIR spectroscopic approach. The D75H mutant is catalytically inactive, whereas the more conservative D75E substitution has quinol oxidase activity equal to that of the wild-type enzyme. Electrochemically induced FTIR difference spectra of the inactive D75H mutant enzyme show a clear decrease in the spectroscopic region characteristic of protonated aspartates and glutamates. Strong variations in the amide I region of the FTIR difference spectrum, however, reflect a more general perturbation due to this mutation of both the protein and the bound quinone. Electrochemically induced FTIR difference spectra on the highly conservative D75E mutant enzyme show a shift from 1734 to 1750 cm(-1) in direct comparison to wild type. After H/D exchange, the mode at 1750 cm(-1) shifts to 1735 cm(-1). These modes, concomitant with the reduced state of the enzyme, can be assigned to the nu(C=O) vibrational mode of protonated D75 and E75, respectively. In the spectroscopic region where signals for deprotonated acidic groups are expected, band shifts for the nu(COO(-))(s/as) modes from 1563 to 1554-1539 cm(-1) and from 1315 to 1336 cm(-1), respectively, are found for the oxidized enzyme. These signals indicate that D75 (or E75 in the mutant) is deprotonated in the oxidized form of cytochrome bo(3) and is protonated upon full reduction of the enzyme. It is suggested that upon reduction of the bound ubiquinone at the high affinity site, D75 takes up a proton, possibly sharing it with ubiquinol.


Subject(s)
Aspartic Acid/metabolism , Cytochromes/metabolism , Escherichia coli/enzymology , Protons , Ubiquinone/analogs & derivatives , Ubiquinone/metabolism , Amides , Aspartic Acid/genetics , Binding Sites/genetics , Cytochrome b Group , Cytochromes/genetics , Electrochemistry , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Escherichia coli/genetics , Escherichia coli Proteins , Glutamic Acid/genetics , Glutamic Acid/metabolism , Histidine/genetics , Mutagenesis, Site-Directed , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared/methods
8.
Biochim Biophys Acta ; 1459(2-3): 305-9, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-11004444

ABSTRACT

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of -80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.


Subject(s)
NADH, NADPH Oxidoreductases/chemistry , Electrochemistry , Electron Spin Resonance Spectroscopy , Electron Transport Complex I , Escherichia coli , Iron-Sulfur Proteins/chemistry , Mitochondria/chemistry , Neurospora crassa , Oxidation-Reduction , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared
9.
Biochemistry ; 39(35): 10884-91, 2000 Sep 05.
Article in English | MEDLINE | ID: mdl-10978175

ABSTRACT

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. It couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to nine iron-sulfur (FeS) clusters participate in the redox reaction. So far, complex I has been described mainly by means of EPR- and UV-vis spectroscopy. Here, we report for the first time an infrared spectroscopic characterization of complex I. Electrochemically induced FT-IR difference spectra of complex I from Escherichia coli and of the NADH dehydrogenase fragment of this complex were obtained for critical potential steps. The spectral contributions of the FMN in both preparations were derived from a comparison using model compounds and turned out to be unexpectedly small. Furthermore, the FT-IR difference spectra reveal that the redox transitions of the FMN and of the FeS clusters induce strong reorganizations of the polypeptide backbone. Additional signals in the spectra of complex I reflect contributions induced by the redox transition of the high-potential FeS cluster N2 which is not present in the NADH dehydrogenase fragment. Part of these signals are attributed to the reorganization of protonated/deprotonated Asp or Glu side chains. On the basis of these data we discuss the role of N2 for proton translocation of complex I.


Subject(s)
Aspartic Acid/chemistry , Escherichia coli/enzymology , Glutamic Acid/chemistry , Iron-Sulfur Proteins/chemistry , NADH, NADPH Oxidoreductases/chemistry , Proton Pumps/chemistry , Alkanesulfonic Acids/chemistry , Aspartic Acid/metabolism , Buffers , Electrochemistry , Electron Transport Complex I , Glutamic Acid/metabolism , Iron-Sulfur Proteins/metabolism , Models, Chemical , Morpholines/chemistry , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Proton Pumps/metabolism , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared/methods
10.
Biochemistry ; 39(6): 1356-63, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10684616

ABSTRACT

By specific (13)C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates [Behr, J., Hellwig, P., Mäntele, W., and Michel, H. (1998) Biochemistry 37, 7400-7406]. To attribute these signals to the individual propionates, we have constructed seven cytochrome coxidase variants using site-directed mutagenesis of subunit I. The mutant enzymes W87Y, W87F, W164F, H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, proton pumping activity, and Vis and FTIR spectroscopy. Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared. Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochromic oxidase. The ring D propionate of heme a(3) might undergo conformational changes or, less likely, act as a proton donor.


Subject(s)
Electron Transport Complex IV/chemistry , Electron Transport Complex IV/genetics , Heme/analogs & derivatives , Heme/chemistry , Paracoccus denitrificans/enzymology , Paracoccus denitrificans/genetics , Amino Acid Substitution/genetics , Arginine/genetics , Electron Transport Complex IV/metabolism , Heme/genetics , Heme/metabolism , Histidine/genetics , Hydrogen Bonding , Mutagenesis, Site-Directed , Oxidation-Reduction , Propionates/chemistry , Propionates/metabolism , Recombinant Proteins/chemistry , Spectroscopy, Fourier Transform Infrared , Tryptophan/genetics , Tyrosine/genetics
11.
FEBS Lett ; 463(1-2): 169-74, 1999 Dec 10.
Article in English | MEDLINE | ID: mdl-10601661

ABSTRACT

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875-12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.


Subject(s)
Phenylalanine/chemistry , Photosynthetic Reaction Center Complex Proteins/chemistry , Rhodobacter sphaeroides/chemistry , Tyrosine/chemistry , Crystallography, X-Ray , Hydrogen Bonding , Light-Harvesting Protein Complexes , Models, Molecular , Mutagenesis, Site-Directed , Oxidation-Reduction , Photosynthetic Reaction Center Complex Proteins/genetics , Rhodobacter sphaeroides/genetics , Spectroscopy, Fourier Transform Infrared
12.
J Biol Chem ; 274(53): 37974-81, 1999 Dec 31.
Article in English | MEDLINE | ID: mdl-10608865

ABSTRACT

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.


Subject(s)
Arginine/genetics , Electron Transport Complex IV/metabolism , Heme/chemistry , Paracoccus denitrificans/enzymology , Chromatography, High Pressure Liquid , Electron Transport , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/genetics , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Oxidation-Reduction , Spectrum Analysis
13.
Biochemistry ; 38(44): 14683-9, 1999 Nov 02.
Article in English | MEDLINE | ID: mdl-10545194

ABSTRACT

In this study we present the infrared spectroscopic characterization of the bound ubiquinone in cytochrome bo(3) from Escherichia coli. Electrochemically induced Fourier transform infrared (FTIR) difference spectra of DeltaUbiA (an oxidase devoid of bound ubiquinone) and DeltaUbiA reconstituted with ubiquinone 2 and with isotopically labeled ubiquinone 2, where (13)C was introduced either at the 1- or at the 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone bound to the discussed high-affinity binding site (Q(H)) are compared to those from the synthetic quinones in solution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm(-)(1), with both carbonyls similarly contributing. The FTIR spectra of DeltaUbiA reconstituted with the labeled quinones indicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutral quinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of the vibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm(-)(1). Only negligible shifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone. Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIR difference spectra. In particular, difference signals at 1640-1633 cm(-)(1) and 1700-1670 cm(-)(1) indicate variations of beta-sheet secondary structure elements and loops, bands at 1706 and 1678 cm(-)(1) are tentatively attributed to individual amino acids, and a difference signal a 1540 cm(-)(1) is discussed to reflect an influence on C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentative assignments are presented and discussed. The (13)C labeling experiments allow the assignment of the vibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra of wild-type bo(3).


Subject(s)
Cytochromes/chemistry , Ubiquinone/chemistry , Binding Sites , Carbon Isotopes , Cytochrome b Group , Electrochemistry , Escherichia coli/chemistry , Escherichia coli Proteins , Hydrogen Bonding , Oxidation-Reduction , Protein Conformation , Spectroscopy, Fourier Transform Infrared , Vibration
14.
FEBS Lett ; 458(2): 83-6, 1999 Sep 17.
Article in English | MEDLINE | ID: mdl-10481041

ABSTRACT

The redox dependent changes in the cytochrome c oxidase from bovine heart were studied with a combined electrochemical and FT-IR spectroscopic approach. A direct comparison to the electrochemically induced FT-IR difference spectra of the cytochrome c oxidase from Paracoccus denitrificans reveals differences in the structure and intensity of vibrational modes. These differences are partially attributed to interactions of subunits influencing the heme and protein modes. In the spectral regions characteristic for v(C=O) and v(COO-)s/as modes of protonated and deprotonated Asp and Glu residues, additional signals at 1736, 1602 and 1588 cm-1 are observed. On this basis, the possible involvement of Asp-51, a residue specifically conserved in mammalian oxidase and previously proposed to show redox depended conformational changes in the respective X-ray structures, is critically discussed.


Subject(s)
Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Myocardium/enzymology , Paracoccus denitrificans/enzymology , Animals , Cattle , Deuterium Oxide/chemistry , Electrochemistry , Oxidation-Reduction , Protein Conformation , Protons , Spectroscopy, Fourier Transform Infrared , Structure-Activity Relationship , Water/chemistry
15.
Biochemistry ; 38(30): 9648-58, 1999 Jul 27.
Article in English | MEDLINE | ID: mdl-10423243

ABSTRACT

The ba3 cytochrome c oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS, and FTIR spectroscopic approach. Oxidative electrochemical redox titrations yielded midpoint potentials of Em1= -0.02 +/- 0.01 V and Em2 = 0.16 +/- 0.04 V for heme b and Em1 = 0.13 +/- 0.04 V and Em2 = 0.22 +/- 0.03 V for heme a(3) (vs Ag/AgCl/3 M KCl). Fully reversible electrochemically induced UV/VIS and FTIR difference spectra were obtained for the full potential step from -0. 5 to 0.5 V as well as for the critical potential steps from -0.5 to 0.1 V (heme b is fully oxidized and heme a3 remains essentially reduced) and from 0.1 to 0.5 V (heme b remains oxidized and heme a3 becomes oxidized). The difference spectra thus allow to us distinguish modes coupled to heme b and heme a3. Analogous difference spectra were obtained for the enzyme in D2O buffer for additional assignments. The FTIR difference spectra reveal the reorganization of the polypeptide backbone, perturbations of single amino acids and of hemes b and a3 upon electron transfer to/from the four redox-active centers heme b and a3, as well as CuB and CuA. Proton transfer coupled to redox transitions can be expected to manifest in the spectra. Tentative assignments of heme vibrational modes, of individual amino acids, and of secondary structure elements are presented. Aspects of the uncommon electrochemical and spectroscopic properties of the ba3 oxidase from T. thermophilus are discussed.


Subject(s)
Cytochrome b Group/chemistry , Electron Transport Complex IV/chemistry , Thermus thermophilus/enzymology , Arginine/chemistry , Aspartic Acid/chemistry , Buffers , Deuterium Oxide/chemistry , Electrochemistry , Glutamic Acid/chemistry , Heme/analogs & derivatives , Heme/chemistry , Lysine/chemistry , Oxidation-Reduction , Peptides/chemistry , Phosphates , Porphyrins/chemistry , Potentiometry , Propionates/chemistry , Protein Conformation , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Tyrosine/chemistry , Water/chemistry
16.
Biochemistry ; 38(23): 7565-71, 1999 Jun 08.
Article in English | MEDLINE | ID: mdl-10360954

ABSTRACT

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction. At 84 K, nonreversible transfer of CO from heme a3 to CuB was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes. Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature. Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme. FT-IR difference spectra show band features similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes induced by electron and coupled proton transfer. These signals disappear when the temperature is lowered to 84 K. At 268 K, a difference signal at 1746 cm-1 is observed which is shifted by 6 cm-1 to 1740 cm-1 in 2H2O. The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.


Subject(s)
Carbon Monoxide/chemistry , Electron Transport Complex IV/chemistry , Paracoccus denitrificans/enzymology , Electron Transport , Electron Transport Complex IV/genetics , Glutamic Acid/genetics , Glutamine/genetics , Mutagenesis, Site-Directed , Oxidation-Reduction , Photolysis , Protons , Spectroscopy, Fourier Transform Infrared , Temperature , Time Factors
17.
Biochemistry ; 38(6): 1685-94, 1999 Feb 09.
Article in English | MEDLINE | ID: mdl-10026246

ABSTRACT

Cytochrome c oxidase from Paracoccus denitrificans was studied with a combined electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectroscopic approach. Global fit analysis of oxidative electrochemical redox titrations was used to separate the spectral contributions coupled to heme a and a3 redox transitions, respectively. Simultaneous adjustment of the midpoint potentials and of the amplitudes for a user-defined number of redox components (here heme a and a3) at all wavelengths in the UV/vis (900-400 nm) and at all wavenumbers in the infrared (1800-1250 cm-1) yielded difference spectra for the number of redox potentials selected. With an assumption of two redox components, two spectra for the redox potential at -0.03 +/- 0.01 V and 0.22 +/- 0.04 V (quoted vs Ag/AgCl) were obtained. The method used here allows the separation of the heme signals from the electrochemically induced visible difference spectra of native cytochrome c oxidase without the addition of any inhibitors. The separated heme a and a3 UV/vis difference spectra essentially correspond to spectra obtained for high/low-spin and 5/6-coordinated heme a/a3 model compounds presented by Babcock [(1988) in Biological Applications of Resonance Raman Spectroscopy (Spiro, T., Ed.) Wiley and Sons, New York]. Single-component Fourier transform infrared (FTIR) difference spectra were calculated for both hemes on the basis of these fits, thus revealing contributions from the reorganization of the polypeptide backbone, from the hemes, and from single amino acids upon electron transfer of the cofactors (heme a/a3, CuA, and CuB), as well from coupled processes such as proton transfer. A tentative assignment of heme vibrational modes is presented and the assignment of the signals to the reorganization of the polypeptide backbone and to perturbations of single amino acids, in particular Asp, Glu, Arg, or Tyr, is discussed.


Subject(s)
Electron Transport Complex IV/chemistry , Heme/analogs & derivatives , Paracoccus denitrificans/enzymology , Arginine/chemistry , Aspartic Acid/chemistry , Electrochemistry , Glutamic Acid/chemistry , Heme/chemistry , Molecular Conformation , Oxidation-Reduction , Porphyrins/chemistry , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Tyrosine/chemistry
18.
Biochim Biophys Acta ; 1409(2): 107-12, 1998 Dec 01.
Article in English | MEDLINE | ID: mdl-9838069

ABSTRACT

In order to study the role of subunits III and IV of the cytochrome c oxidase from P. denitrificans for electron and proton transfer, electrochemically induced FT-IR difference spectra of the two- and of the four-subunit enzyme have been compared. These spectra reflect the alterations in the protein upon electron and proton transfer. Since the spectra are essentially identical, they clearly indicate that the additional subunits III and IV do not contribute to the FT-IR difference spectra of the four-subunit oxidase. Subunits III and IV are thus not involved in the reorganization of the polypeptide backbone and of single amino acids upon electron transfer and coupled proton transfer observed in the difference spectra in addition to heme contributions. The subtle differences between the FT-IR difference spectra that are attributed to the influence of protein-protein interactions between the subunits are discussed.


Subject(s)
Electron Transport Complex IV/chemistry , Soil Microbiology , Binding Sites , Electrochemistry , Oxidation-Reduction , Protein Conformation , Spectroscopy, Fourier Transform Infrared
19.
Biochemistry ; 37(20): 7390-9, 1998 May 19.
Article in English | MEDLINE | ID: mdl-9585553

ABSTRACT

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620-1680 cm-1) and amide II (ca. 1560-1540 cm-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mäntele, W. (1996) FEBS Lett. 385, 53-57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.


Subject(s)
Electron Transport Complex IV/chemistry , Glutamic Acid/chemistry , Paracoccus denitrificans/chemistry , Asparagine/genetics , Aspartic Acid/genetics , Deuterium Oxide/chemistry , Electron Transport Complex IV/genetics , Glutamic Acid/genetics , Glutamine/genetics , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Oxidation-Reduction , Paracoccus denitrificans/genetics , Serine/genetics , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared
20.
Biochemistry ; 37(20): 7400-6, 1998 May 19.
Article in English | MEDLINE | ID: mdl-9585554

ABSTRACT

Specific isotope labeling at the carboxyl groups of the four heme propionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redox Fourier transform infrared (FTIR) difference spectra of this enzyme. For this purpose, the hemA gene of the P. denitrificans strain PD1222, coding for 5-aminolevulinate synthase, was deleted by partial replacement with a kanamycin resistance cartridge, resulting in a stable 5-aminolevulinic acid (ALA) auxotrophy. Normal growth of this deficient strain and cytochrome c oxidase yield comparable to that of P. dentrificans wild-type strain PD1222 could be obtained by supplementation with 0.1 mM ALA in the growth medium. Visible spectra and reduced-minus-oxidized FTIR spectra showed that the purified cytochrome c oxidase had spectral characteristics identical to those of the wild-type enzyme. The decrease of a negative signal at 1676 cm-1 in the reduced-minus-oxidized FTIR difference spectra of the 13C-labeled cytochrome c oxidase in comparison to those of the unlabeled protein allowed the assignment of this signal to a COOH vibration mode of at least one of the four heme propionates. Moreover, a negative band at approximately 1570 cm-1 shifted to smaller wavenumbers in the spectra of the 13C-labeled enzyme in comparison to the spectra of the unlabeled enzyme and was thus assigned to contributions from an antisymmetric COO- mode of one or more of the four heme propionates. Additionally, a positive signal at 1538 cm-1 shifted to approximately 1500 cm-1 in the spectra of the isotopically labeled protein and was therefore assigned to at least one antisymmetric COO- mode of the heme propionates. A negative signal at 1390 cm-1, which has been shifted to 1360 cm-1 in the spectra of the 13C-labeled enzyme, is due to a symmetric COO- mode from at least one heme propionate. These results suggest that at least two of the four heme propionates in cytochrome c oxidase undergo significant vibrational changes upon reduction of the enzyme, either by protonation/deprotonation or by environmental changes.


Subject(s)
Electron Transport Complex IV/chemistry , Heme/chemistry , Isotope Labeling , Paracoccus denitrificans/enzymology , 5-Aminolevulinate Synthetase/genetics , Carbon Isotopes , Cytochrome-c Oxidase Deficiency , Electron Transport Complex IV/genetics , Electron Transport Complex IV/isolation & purification , Electron Transport Complex IV/metabolism , Gene Deletion , Heme/metabolism , Mutagenesis, Site-Directed , Oxidation-Reduction , Paracoccus denitrificans/genetics , Spectroscopy, Fourier Transform Infrared
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