Electrochemical, FTIR, and UV/VIS spectroscopic properties of the ba(3) oxidase from Thermus thermophilus.

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.     

Electrochemical, FT-IR and UV/VIS spectroscopic properties of the caa3 oxidase from T. thermophilus.

The caa3-oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS and Fourier-transform infrared (FT-IR) spectroscopic approach. In this oxidase the electron donor, cytochrome c, is covalently bound to subunit II of the cytochrome c oxidase. Oxidative electrochemical redox titrations in the visible spectral range yielded a midpoint potential of -0.01 +/- 0.01 V (vs. Ag/AgCl/3m KCl, 0.218 V vs. SHE') for the heme c. This potential differs for about 50 mV from the midpoint potential of isolated cytochrome c, indicating the possible shifts of the cytochrome c potential when bound to cytochrome c oxidase. For the signals where the hemes a and a3 contribute, three potentials, = -0.075 V +/- 0.01 V, Em2 = 0.04 V +/- 0.01 V and Em3 = 0.17 V +/- 0.02 V (0.133, 0.248 and 0.378 V vs. SHE', respectively) could be obtained. Potential titrations after addition of the inhibitor cyanide yielded a midpoint potential of -0.22 V +/- 0.01 V for heme a3-CN- and of Em2 = 0.00 V +/- 0.02 V and Em3 = 0.17 V +/- 0.02 V for heme a (-0.012 V, 0.208 V and 0.378 V vs. SHE', respectively). The three phases of the potential-dependent development of the difference signals can be attributed to the cooperativity between the hemes a, a3 and the CuB center, showing typical behavior for cytochrome c oxidases. A stronger cooperativity of CuB is discussed to reflect the modulation of the enzyme to the different key residues involved in proton pumping. We thus studied the FT-IR spectroscopic properties of this enzyme to identify alternative protonatable sites. The vibrational modes of a protonated aspartic or glutamic acid at 1714 cm-1 concomitant with the reduced form of the protein can be identified, a mode which is not present for other cytochrome c oxidases. Furthermore modes at positions characteristic for tyrosine vibrations have been identified. Electrochemically induced FT-IR difference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the nu(C=O) modes, which reflect the high degree of similarity of heme a3 to other typical heme copper oxidases. A comparison with previously studied cytochrome c oxidases is presented and on this basis the contributions of the reorganization of the polypeptide backbone, of individual amino acids and of the hemes c, a and a3 upon electron transfer to/from the redox active centers discussed.     

Redox titration of all electron carriers of cytochrome c oxidase by Fourier transform infrared spectroscopy

Electrochemical redox titrations of cytochrome c oxidase from Paraccocus denitrificans were performed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The majority of the differential infrared absorption features may be divided into four groups, which correlate with the redox transitions of the four redox centers of the enzyme. Infrared spectroscopy has the advantage of allowing one to measure independent alterations in redox centers, which are not well separated, or even observed, by other spectroscopic techniques. We found 12 infrared bands that titrated with the highest observed midpoint redox potential (E(m) = 412 mV at pH 6.5) and which had a pH dependence of 52 mV per pH unit in the alkaline region. These bands were assigned to be linked to the Cu(B) center. We assigned bands to the Cu(A) center that showed a pH-independent E(m) of 250 mV. Two other groups of infrared differential bands reflected redox transitions of the two heme groups and showed a more complex behavior. Each of them included two parts, corresponding to high- and low-potential redox transitions. For the bands representing heme a, the ratio of high- to low-potential components was ca. 3:2; for heme a(3) this ratio was ca. 2:3. Taking into account the redox interactions between the hemes, these ratios yielded a difference in E(m) of 9 mV between the hemes (359 mV for heme a; 350 mV for heme a(3) at pH 8.0). The extent of the redox interaction between the hemes (-115 mV at pH 8.0) was found to be pH-dependent. The pH dependence of the E(m) values for the two hemes was the same and about two times smaller than the theoretical one, suggesting that an acid/base group binds a proton upon reduction of either heme. The applied approach allowed assignment of infrared bands in each of the four groups to vibrations of the hemes, ligands of the redox centers, amino acid residues, and/or protein backbone. For example, the well-known band shift at 1737/1746 cm(-)(1) corresponding to the protonated glutamic acid E278 correlated with oxidoreduction of heme a.     

Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center

Cytochrome bd is a quinol oxidase from Escherichia coli, which is optimally expressed under microaerophilic growth conditions. The enzyme catalyzes the two-electron oxidation of either ubiquinol or menaquinol in the membrane and scavenges O2 at low concentrations, reducing it to water. Previous work has shown that, although cytochrome bd does not pump protons, turnover is coupled to the generation of a proton motive force. The generation of a proton electrochemical gradient results from the release of protons from the oxidation of quinol to the periplasm and the uptake of protons used to form H2O from the cytoplasm. Because the active site has been shown to be located near the periplasmic side of the membrane, a proton channel must facilitate the delivery of protons from the cytoplasm to the site of water formation. Two conserved glutamic acid residues, E107 and E99, are located in transmembrane helix III in subunit I and have been proposed to form part of this putative proton channel. In the current work, it is shown that mutations in either of these residues results in the loss of quinol oxidase activity and can result in the loss of the two hemes at the active site, hemes d and b595. One mutant, E107Q, while being totally inactive, retains the hemes. Fourier transform infrared (FTIR) redox difference spectroscopy has identified absorption bands from the COOH group of E107. The data show that E107 is protonated at pH 7.6 and that it is perturbed by the reduction of the heme d/heme b595 binuclear center at the active site. In contrast, mutation of an acidic residue known to be at or near the quinol-binding site (E257A) also inactivates the enzyme but has no substantial influence on the FTIR redox difference spectrum. Mutagenesis shows that there are several acidic residues, including E99 and E107 as well as D29 (in CydB), which are important for the assembly or stability of the heme d/heme b595 active site.     

Attenuated total reflection Fourier transform infrared studies of redox changes in bovine cytochrome c oxidase: resolution of the redox Fourier transform infrared difference spectrum of heme a(3).

Attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy has been performed on samples of bovine cytochrome c oxidase that have been deposited as a thin film on the surface of a silicon microprism. The technique has several advantages over transmission methods in terms of amount of material required, the time required to reach sufficient optical stability, and the range of reactants that can be repetitively added and removed. The ATR-FTIR method has been used to record redox difference spectra of cytochrome c oxidase in the unligated and cyanide-ligated states. By subtraction of the spectra, the redox FTIR difference spectrum of heme a(3) can be resolved from those of the other metal centers. This difference spectrum is compared with available vibrational and Raman data on homologous oxidases and on heme A model compounds.     

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic redox behavior of the heme centers of cbb3 heme-copper oxygen reductase from Bradyrhizobium japonicum

A comprehensive study of the thermodynamic redox behavior of the hemes from the cbb3 oxygen reductase from Bradyrhizobium japonicum was performed. This enzyme is a member of the C-type heme-copper oxygen reductase superfamily and has three subunits with six redox centers: four low-spin hemes and a high-spin heme and one copper ion, composing the site where oxygen is reduced. In this analysis, the visible spectra and redox properties of the five heme centers were deconvoluted. Their redox profiles and the pH dependence of the midpoint reduction potentials (redox-Bohr effect) were investigated. The reference reduction potentials (defined for a state where all centers are reduced) and homotropic interaction potentials were determined in the framework of a model of pairwise interacting redox centers. At pH 7.7, the reference reduction potentials for the three hemes c are 390, 300, and 220 mV, with low interaction potentials between them, weaker than -15 mV. For hemes b and b3, reference reduction potentials of 375 and 290 mV, respectively, were obtained; these two redox centers show an interaction potential weaker than -60 mV. The midpoint reduction potentials of all five hemes are pH-dependent. The study of these thermodynamic parameters is important in understanding the coupling mechanism of the redox and chemical processes during oxygen reduction. The analysis of the thermodynamic redox behavior of the cbb3 oxygen reductase contributes to the investigation of the mechanism of electron transfer and proton translocation by heme-copper oxygen reductases in general and indicates a thermodynamic coupling for the electron and proton transfer mechanisms.     

Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a₃, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa₃-type cytochrome c oxidase consists of overlapping bands of the hemes a²⁺ and a₃²⁺. It shows a peak at ～444 nm and a distinct shoulder at ～425 nm. However, attribution of individual spectral lineshapes to hemes a²⁺ and a₃²⁺ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a²⁺ and a₃²⁺ using two approaches. First, we reconstructed bovine CcO heme a²⁺ spectrum using a selective Ca²⁺-induced spectral shift of the heme a²⁺. Second, we investigated photobleaching of the reduced Thermus thermophilus ba₃- and bovine aa₃-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B_0x-, B_0y-transitions of both reduced hemes. The heme a²⁺ spectrum is shifted to the red relative to heme a₃²⁺ spectrum. The ～425 nm shoulder is mostly attributed to heme a₃²⁺.     

Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase

The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.     

Heme/heme redox interaction and resolution of individual optical absorption spectra of the hemes in cytochrome bd from Escherichia coli

Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. It contains three hemes, b₅₅₈, b₅₉₅, and d. The detailed spectroelectrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b₅₅₈ and high-spin heme b₅₉₅, whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The α- and β-band reduced-minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, M.J. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051-1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of “cytochrome a₁” as cytochrome b₅₉₅, Biochemistry 25 (1986) 2314-2321.). The Soret band spectra show λ_max = 429.5 nm, λ_min ≈ 413 nm (heme b₅₅₈), λ_max = 439 nm, λ_min ≈ 400 ± 1 nm (heme b₅₉₅), and λ_max = 430 nm, λ_min = 405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/α (ΔA₄₃₀:ΔA₆₂₉) ratio for heme d is 1.6.     

Characterization of the high-spin heme x in the cytochrome b6f complex of oxygenic photosynthesis

X-ray structures at 3.0-3.1 A resolution of the cytochrome b(6) f complex from the cyanobacterium Mastigocladus laminosus [Kurisu, G., Zhang, H., Smith, J. L., and Cramer, W. A. (2003) Science 302, 1009-1014] and the green alga Chlamydomonas reinhardtii [Stroebel, D., Choquet, Y., Popot, J.-L., and Picot, D. (2003) Nature 426, 413-418] showed the presence of a unique heme, hemex, that is covalently linked by a single thioether bond to a Cys residue (Cys35) on the electrochemically negative (n) side of the cytochrome b(6) polypeptide. Heme x faces the intermonomer quinone exchange cavity. The only axial ligand associated with this heme is a H(2)O or OH(-) that is H-bonded to the propionate of the stromal side heme b(n), showing that it is pentacoordinate. The spectral properties of this heme were hardly defined at the time of the structure determination. The pyridine hemochromagen redox difference spectrum for heme x covalently bound to the cytochrome b polypeptide isolated from SDS-PAGE displays a low-amplitude broad spectrum with a peak at 553 nm, similar to that of other hemes with a single thioether linkage. The binding of CO and a hydrophobic cyanide analogue, butyl isocyanide, to dithionite-reduced b(6) f complex perturbs and significantly shifts the redox difference visible spectrum. Together with EPR spectra displaying g values of the oxidized complex of 6.7 and 7.4, heme x is defined as a ferric high-spin heme in a rhombic environment. In addition to a possible function in photosystem I-linked cyclic electron transport, the five-coordinate state implies that there is at least one more function of heme x that is related to axial binding of a physiological ligand.     

Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans. Evidence from FTIR difference spectroscopy and site-directed mutagenesis

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., M?ntele, 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.     

M?ssbauer characterization of the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Spectral deconvolution of the heme components.

M?ssbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic M?ssbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic M?ssbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic M?ssbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.     

A novel double heme substitution produces a functional bo3 variant of the quinol oxidase aa3 of Bacillus cereus. Purification and paratial characterization

A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa3 and caa3. The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg-1 protein. The enzyme was composed of two subunits with an Mr of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa3 oxidase. Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B. cereus aa3 oxidase. The purified bo3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent Km of 498 microM, a Vmax of 21 micromol of O2 min-1mg-1, and a calculated turnover of 55 s-1. The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I50 of 24 and 300 microM, respectively. Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome. This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites.     

Redox-induced transitions in bovine cytochrome bc1 complex studied by perfusion-induced ATR-FTIR spectroscopy

Redox transitions in a film of detergent-purified bovine cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The technique provides a flexible method for generating redox-induced IR changes of components of bovine cytochrome bc(1) complex at a high signal:noise ratio. These IR redox difference spectra arise from perturbations of prosthetic groups and surrounding protein. Visible difference spectra were recorded synchronously using a light beam reflected from the exposed prism surface and provided a quantitative means of determining the redox transitions that were occurring. IR and visible redox difference spectra of iron-sulfur protein/cytochrome c(1), heme b(H), and heme b(L) were separated by selective reduction and/or oxidation that extends published data on the homologous bacterial enzyme. Several bands could be tentatively assigned to redox-sensitive modes of hemes and ubiquinone and changes in the surrounding protein by comparison with available data for bacterial bc(1) complex, other related heme proteins, and model compounds. Some tentative assignments of further signals to specific amino acids are made on the basis of known crystal structures.     

Circular dichroism spectra of cytochrome c oxidase

Circular dichroism spectra of bovine heart aa(3)-type cytochrome c oxidase have been studied with a major focus on the Soret band π → π* transitions, B(0(x,y)), in the two iron porphyrin groups of the enzyme. The spectra of the fully reduced and fully oxidized enzyme as well as of its carbon monoxide and cyanide complexes have been explored. In addition, CD spectra of the reduced and oxidized ba(3)-type cytochrome c oxidase from Thermus thermophilus were recorded for comparison. An attempt is made to interpret the CD spectra of cytochrome c oxidase with the aid of a classical model of dipole-dipole coupled oscillators taking advantage of the known 3D crystal structure of the enzyme. Simultaneous modeling of the CD and absorption spectra shows that in the bovine oxidase, the dipole-dipole interactions between the hemes a and a(3), although contributing significantly, cannot account either for the lineshape or the magnitude of the experimental spectra. However, adding the interactions of the hemes with 22 aromatic amino acid residues located within 12 ? from either of the two heme groups can be used to model the CD curves for the fully reduced and fully oxidized oxidase with reasonable accuracy. Interaction of the hemes with the peptide bond transition dipoles is found to be insignificant. The modeling indicates that the CD spectra of cytochrome oxidase in both the reduced and oxidized states are influenced significantly by interaction with Tyr244 in the oxygen-reducing center of the enzyme. Hence, CD spectroscopy may provide a useful tool for monitoring the redox/ionization state of this residue. The modeling confirms wide energy splitting of the orthogonal B(x) and B(y) transitions in the porphyrin ring of heme a.     

Identification of novel hemes generated by heme A synthase: evidence for two successive monooxygenase reactions

Heme A, an obligatory cofactor in eukaryotic cytochrome c oxidase, is produced from heme B (protoheme) via two enzymatic reactions catalyzed by heme O synthase and heme A synthase. Heme O synthase is responsible for the addition of a farnesyl moiety, while heme A synthase catalyzes the oxidation of a methyl substituent to an aldehyde. We have cloned the heme O synthase and heme A synthase genes from Bacillus subtilis (ctaB and ctaA) and overexpressed them in Escherichia coli to probe the oxidative mechanism of heme A synthase. Because E. coli does not naturally produce or utilize heme A, this strategy effectively decoupled heme A biosynthesis from the native electron transfer pathway and heme A transport, allowing us to observe two previously unidentified hemes. We utilized HPLC, UV/visible spectroscopy, and tandem mass spectrometry to identify these novel hemes as derivatives of heme O containing an alcohol or a carboxylate moiety at position C8 on pyrrole ring D. We interpret these derivatives to be the putative alcohol intermediate and an overoxidized byproduct of heme A synthase. Because we have shown that all hemes produced by heme A synthase require O(2) for their synthesis, we propose that heme A synthase catalyzes the oxidation of the C8 methyl to an aldehyde group via two discrete monooxygenase reactions.     

Electrochemically induced FT-IR difference spectra of the two- and four-subunit cytochrome c oxidase from P. denitrificans reveal identical conformational changes upon redox transitions

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.     

Full assignment of heme redox potentials of cytochrome c3 of D. vulgaris Miyazaki F by 1H-NMR.

Site-specific heme assignment of the 1H-NMR spectrum of cytochrome c3 of D. vulgaris Miyazaki F, a tetraheme protein, was established. The major reduction of the heme turned out to take place in the order of hemes I, III, IV and II (numbering in the crystal structure). The hemes with the smallest and greatest solvent accessibility were reduced at the highest and lowest potentials on average, respectively. A cooperative interheme interaction was attributed to a pair of the closest hemes, namely, hemes III and IV. This assignment can provide the physiochemical basis for the elucidation of electron transfer of this protein.