Redox-induced protein structural changes in cytochrome bo revealed by Fourier transform infrared spectroscopy and [13C]Tyr labeling

Cytochrome bo is a heme-copper terminal ubiquinol oxidase of Escherichia coli under highly aerated growth conditions. Tyr-288 present at the end of the K-channel forms a Cepsilon-Nepsilon covalent bond with one of the Cu(B) ligand histidines and has been proposed to be an acid-base catalyst essential for the O-O bond cleavage at the Oxy-to-P transition of the dioxygen reduction cycle (Uchida, T., Mogi, T., and Kitagawa, T. (2000) Biochemistry 39, 6669-6678). To probe structural changes at tyrosine residues, we examined redox difference Fourier transform infrared difference spectra of the wild-type enzyme in which either L-[1-13C]Tyr or L-[4-13C]Tyr has been biosynthetically incorporated in the tyrosine auxotroph. Spectral comparison between [1-13C]Tyr-labeled and unlabeled proteins indicated that substitution of the main chain carbonyl of a Tyr residue(s) significantly affected changes in the amide-I (approximately 1620-1680 cm(-1)) and -II ( approximately 1540-1560 cm(-1)) regions. In contrast, spectral comparison between [4-13C]Tyr-labeled and unlabeled proteins showed only negligible changes, which was the case for both the pulsed and the resting forms. Thus, protonation of an OH group of tyrosines including Tyr-288 in the vicinity of the heme o-Cu(B) binuclear center was not detected at pH 7.4 upon full reduction of cytochrome bo. Redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal centers and may be related to switching of the K-channel operative at the reductive phase to D-channel at the oxidative phase of the dioxygen reduction cycle via conformational changes in the middle of helix VI.     

Probing protonation/deprotonation of tyrosine residues in cytochrome ba3 oxidase from Thermus thermophilus by time-resolved step-scan Fourier transform infrared spectroscopy

Elucidating the properties of the heme Fe-Cu(B) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν(7a)(CO) of the protonated form of Tyr residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). By monitoring the intensity changes of the 1247 and 1301 cm(-1) modes as a function of pH, we measured a pK(a) of 7.8 for the observed tyrosine. The FTIR spectral changes associated with the tyrosine do not belong to Tyr-237 but are attributed to the highly conserved in heme-copper oxidases Tyr-136 and/or Tyr-133 residue (Koutsoupakis, K., Stavrakis, S., Pinakoulaki, E., Soulimane, T., and Varotsis, C. (2002) J. Biol. Chem. 277, 32860-32866). The oxygenation of CO by the mixed-valence form of the enzyme revealed the formation of the ～607 nm P (Fe(IV)=O) species in the pH 6-9 range and the return to the oxidized form without the formation of the 580 nm F form. The data indicate that Tyr-237 is not involved in the proton transfer pathway in the oxygenation of CO by the mixed-valence form of the enzyme. The implication of these results with respect to the role of Tyr-136 and Tyr-133 in proton transfer/gating along with heme a(3) ring D propionate-H(2)O-ring A propionate-Asp-372 site to the exit/output proton channel (H(2)O pool) is discussed.     

pH-dependent structural changes at the Heme-Copper binuclear center of cytochrome c oxidase

The resonance Raman spectra of the aa3 cytochrome c oxidase from Rhodobacter sphaeroides reveal pH-dependent structural changes in the binuclear site at room temperature. The binuclear site, which is the catalytic center of the enzyme, possesses two conformations at neutral pH, assessed from their distinctly different Fe-CO stretching modes in the resonance Raman spectra of the CO complex of the fully reduced enzyme. The two conformations (alpha and beta) interconvert reversibly in the pH 6-9 range with a pKa of 7.4, consistent with Fourier transform infrared spectroscopy measurements done at cryogenic temperatures (D.M. Mitchell, J.P. Sapleigh, A.M.Archer, J.O. Alben, and R.B.Gennis, 1996, Biochemistry 35:9446-9450). It is postulated that the different structures result from a change in the position of the Cu(B) atom with respect to the CO due to the presence of one or more ionizable groups in the vicinity of the binuclear center. The conserved tyrosine residue (Tyr-288 in R. sphaeroides, Tyr-244 in the bovine enzyme) that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu(B) are possible candidates. The existence of an equilibrium between the two conformers at physiological pH and room temperature suggests that the conformers may be functionally involved in enzymatic activity.     

Exposure of bovine cytochrome c oxidase to high triton X-100 or to alkaline conditions causes a dramatic change in the rate of reduction of compound F.

The final step in the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a(3) in compound F, was investigated using a binuclear polypyridine ruthenium complex ([Ru(bipyridine)(2)](2)(1,4-bis[2-(4'-methyl-2, 2'-bipyrid-4-yl)ethenyl]benzene)(PF(6))(4)) as a photoactive reducing agent. In the untreated dimeric enzyme, the rate constant for reduction of compound F decreased from 700 s(-1) to 200 s(-1) as the pH was increased from 7.5 to 9.5. Incubation of dimeric enzyme at pH 10 led to an increase in the rate constant to 1650 s(-1), which was independent of pH between pH 7.4 and 10. This treatment resulted in a decrease in the sedimentation coefficient consistent with the irreversible conversion of the enzyme to a monomeric form. Similar results were obtained when the enzyme was incubated with Triton X-100 at pH 8.0. These treatments, which have traditionally been used to convert dimeric enzyme to monomeric form, have no effect on the steady-state activity. The data indicate that either the conversion of the bovine oxidase to a monomeric form or some structural change coincident with this conversion strongly influences the rate constant of this step in the catalytic cycle, perhaps by influencing the proton access to the heme-copper binuclear center.     

Effects of subunit I mutations on redox-linked conformational changes of the Escherichia coli bo-type ubiquinol oxidase revealed by Fourier-transform infrared spectroscopy.

Cytochrome bo is the heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli, and functions as a redox-coupled proton pump. As an extension to our mutagenesis and Fourier-transform infrared studies on ion pumps, we examined the effects of subunit I mutations on redox-linked protein structural changes in cytochrome bo. Upon photo-reduction in the presence of riboflavin, Y288F and H333A showed profound effects in their peptide backbone vibrations (amide-I and amide-II), probably due to the loss of CuB or replacement of high-spin heme o with heme B. In the frequency region of protonated carboxylic C=O stretching vibrations, negative 1,743 cm-1 and positive 1,720 cm-1 bands were observed in the wild-type; the former shifted to 1,741 cm-1 in E286D but not in other mutants including D135N. This suggests that Glu286 in the D-channel is protonated in the air-oxidized state and undergoes hydrogen bonding changes upon reduction of the redox metal centers. Two pairs of band shifts at 2,566 (+)/2,574 (-) and 2,546 (+)/2,556 (-) cm-1 in all mutants indicate that two cysteine residues not in the vicinity of the metal centers undergo redox-linked hydrogen bonding changes. Cyanide had no effect on the protein structural changes because of the rigid local protein structure around the binuclear center or the presence of a ligand(s) at the binuclear center, and was released from the binuclear center upon reduction. This study establishes that cytochrome bo undergoes unique redox-linked protein structural changes. Localization and time-resolved analysis of the structural changes during dioxygen reduction will facilitate understanding of the molecular mechanism of redox-coupled proton pumping at the atomic level.     

A molecular dynamics study of water chain formation in the proton-conducting K channel of cytochrome c oxidase

The formation of water chains in cytochrome c oxidase (CcO) is studied by molecular dynamics (MD). Focus is on water chains in the K channel that can supply a proton to the binuclear center (the heme a3 Fe/CuB region), the site of O2 reduction. By assessing the presence of chains of any length on a short time scale (0.1 ps), a view of the kinds of chains and their persistence is obtained. Chains from the entry of the channel on the inner membrane to Thr359 (Rhodobacter sphaeroides numbering) are often present but are blocked at that point until a rotation of the Thr359 side chain occurs, permitting formation of chains from Thr359 towards the binuclear center. No continuous hydrogen-bonded water chains are found connecting Thr359 and the binuclear center. Instead, waters hydrogen bond from Thr359 to the hydroxyl of the heme a3 farnesyl and then continue to the binuclear center via Tyr288, which has been identified as a source of a proton for O2 reduction. Three hydrogen-bonded waters are found to be present in the binuclear center after a sufficiently long simulation time. One is ligated to the CuB and could be associated with a water (or hydroxyl) identified in the crystal structure as the fourth ligand of CuB. The water hydrogen-bonded to the hydroxyl of Tyr288 is extremely persistent and well positioned to participate in O2 reduction. The third water is located where O2 is often suggested to reside in mechanistic studies of O2 reduction.     

Fourier transform infrared (FTIR) and step-scan time-resolved FTIR spectroscopies reveal a unique active site in cytochrome caa3 oxidase from Thermus thermophilus

Fourier transform infrared (FTIR) and step-scan time-resolved FTIR difference spectra are reported for the [carbonmonoxy]cytochrome caa(3) from Thermus thermophilus. A major C-O mode of heme a(3) at 1958 cm(-1) and two minor modes at 1967 and 1975 cm(-1) (7:1:1) have been identified at room temperature and remained unchanged in H(2)O/D(2)O exchange. The observed C-O frequencies are 10 cm(-1) higher than those obtained previously at 21 K (Einarsdóttir, O., Killough, P. M., Fee, J. A., and Woodruff, W. H. (1989) J. Biol. Chem. 264, 2405-2408). The time-resolved FTIR data indicate that the transient Cu(B)(1+)-CO complex is formed at room temperature as revealed by the CO stretching mode at 2062 cm(-1). Therefore, the caa(3) enzyme is the only documented member of the heme-copper superfamily whose binuclear center consists of an a(3)-type heme of a beta-form and a Cu(B) atom of an alpha-form. These results illustrate that the properties of the binuclear center in other oxidases resulting in the alpha-form are not required for enzymatic activity. Dissociation of the transient Cu(B)(1+)-CO complex is biphasic. The rate of decay is 2.3 x 10(4) s(-1) (fast phase, 35%) and 36.3 s(-1) (slow phase, 65%). The observed rate of rebinding to heme a(3) is 34.1 s(-1). The implications of these results with respect to the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases are discussed.     

Fourier-transform infrared studies on azide-binding to the binuclear center of the Escherichia coli bo-type ubiquinol oxidase.

Azide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier-transform infrared spectroscopy. Deconvolution analyses of infrared spectra of the azide (14N3)-inhibited air-oxidized form showed a major infrared azide antisymmetric stretching band at 2041 cm(-1). An additional band developed at 2062.5 cm(-1) during a longer incubation. Isotope substitutions with terminally 15N-labelled azides did not show a splitting of the major band, indicating that the geometry of the bound azide is mainly in a bridging configuration between high-spin heme o and CuB. The band at 2062.5 cm(-1) showed clear splittings upon substitution with the terminally 15N-labelled azides, indicating the Cu(2+)B-N=N=N structure. Partial reduction of the oxidase with beta-NADH in the presence of azide caused an appearance of new infrared bands at 2038.5 (major) and 2009 (minor) cm(-1). The former band also showed clear splittings in the presence of the terminally 15N-labelled azides, indicating that reduction of low-spin heme b alters the structure of the binuclear center leading to the Fe(3+)o-N=N=N configuration.     

Electron transfer at the low-spin heme b of cytochrome bo(3) induces an environmental change of the catalytic enhancer glutamic acid-286

Intramolecular proton transfer of heme-copper oxidases is performed via the K- and the transmembrane D-channels. A carboxyl group conserved in a subgroup of heme-copper oxidases, located within the D-channel close to the binuclear center (=glutamic acid-286 in cytochrome bo(3) from Escherichia coli) is essential for proton pumping. Upon electron transfer to the fully oxidized (FO) enzyme, this amino acid has been shown to undergo a cyanide-independent environmental change. The redox-induced environmental transition of glutamic acid-286 is preserved in the site-directed mutant Y288F, which has lost its Cu(B) binding capacity. Furthermore, the mixed-valence (MV) redox state of cytochrome bo(3) (in which Cu(B) and high-spin heme are reduced, whereas the low-spin heme stays oxidized) was prepared by anaerobic exposure of the protein to carbon monoxide. This complex was converted (i) to the FO state by reaction with the caged dioxygen donor mu-peroxo) (mu-hydroxo) bis [bis (bipyridyl) cobalt (III)] and (ii) to the fully reduced (FR) state via caged electron donors; the environmental change of glutamic acid-286 could be observed only upon reduction. Taken together, these results from two different lines of evidence clearly show that the redox transition of the low-spin heme b center alone triggers the change in the chemical environment of this acidic side chain. It is suggested that glutamic acid-286 is a kinetic enhancer of proton translocation, which is energetically favoured in mesophilic oxidases.     

Comparative biological activities of potent active-site analogues of alpha-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several alpha-melanotropin (alpha-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr4]-alpha-MSH4-10-NH2 and Ac-[Tyr4]-alpha-MSH4-11-NH2 were compared with alpha-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr4]-alpha-MSH4-10-NH2 was found to be less active than Ac-[Tyr4]-alpha-MSH4-11-NH2 on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr4]-alpha-MSH4-10-NH2 exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr4]-alpha-MSH4-11-NH2 was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr4]-alpha-MSH4-10-NH2 over Ac-[Tyr4]-alpha-MSH4-11-NH2 on frog melanocytes may be related to the fact that the shorter 4-10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of alpha-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle4]-alpha-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

Design of oxytocin antagonists, which are more selective than atosiban.

We report the solid phase synthesis of four pairs of L- and D-thienylalanine (Thi/D-Thi) position two modified analogues of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly(NH2)9,d (CH2)5[Tyr(Me)2,Thr4]OVT) (A); the Tyr-(NH2)9 analogue of (A), d(CH2)5[Tyr(Me)2,Thr4,Tyr-(NH2)9]OVT (B); the Eda9 analogue (where Eda = ethylenediamine) of (A), d(CH2)5[Tyr(Me)2, Thr4, Eda9]OVT (C); and the retro Tyr10 modified analogue of (C), d(CH2)5[Tyr(Me)2, Thr4, Eda9<--Tyr10]OVT (D). The eight new analogues of A-D are (1) desGly(NH2),d(CH2)5[Thi2,Thr4]OVT, (2) desGly(NH2),d(CH2)5[D-Thi2,Thr4]OVT, (3) d(CH2)5[Thi2, Thr4,Tyr-(NH2)9]OVT, (4) d(CH2)5[D-Thi2,Thr4,Tyr-(NH2)9]OVT (5) d(CH2)5[Thi2,Thr4Eda9]OVT, (6) d(CH2)5[D-Thi2,Thr4,Eda9]OVT, (7) d(CH2) [Thi2,Thr4,Eda9<--Tyr10]OVT, (8) d(CH2),[D-Thi2,Thr4,Eda9<--Tyr10]OVT. We also report the synthesis of (C). Peptides 1-8 and C were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in in vivo vasopressor (V1a receptor) assays and in in vivo antidiuretic (V2 receptor) assays. None of the eight peptides nor C exhibit oxytocic or vasopressor agonism. Peptides 1-8 are extremely weak V2 agonists (antidiuretic activities range from < 0.0005 to 0.20 U/mg). Peptide C is a weak mixed V2 agonist/antagonist. Peptides 1-8 and C exhibit potent in intro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.76 to 8.05). Peptides 1-8 are all OT antagonists in vivo (estimated in vivo anti-OT pA2 values range from 6.54-7.19). With anti-V1a pA2 values of approximately 5-5.80, peptides 1-8 exhibit marked reductions in anti-V1a potencies relative to those of the parent peptides A-D (anti-V1a pA2 range from 6.48 to 7.10) and to l-deamino[D-Tyr(Et)2, Thr4]OVT (Atosiban, trade name Tractocile) (anti-V1a pA2-6.14). Atosiban has recently been approved in Europe for clinical use for the prevention of premature labour (Pharm. J. 264(7-100): 871). Peptides 1-8 exhibit striking gains in in vitro anti-OT/anti-V1a selectivities with respect to the parent peptides A, B, C and D and to Atosiban. Peptides 1-8 exhibit anti-OT (in vitro)/anti-V1a selectivities of 450, 525, 550, 450, approximately 1080, 116, 355, 227 respectively. The corresponding values for A-D and Atosiban are 30, 4.2, 4.3, 2.6 and 37. With the exception of peptide 6, the remaining seven peptides exhibit 3-18-fold gains in anti-OT (in vivo)/anti-V1a selectivity with respect to Atosiban, peptides 1-8 exhibit anti-OT (in vivo)/anti-V1a selectivities of 22, approximately 82, approximately 82, 147, approximately 83, 11, 31 and 42. By comparison, Atosiban exhibits an anti-OT (in vivo)/anti-V1a selectivity = 8. With an estimated in vivo anti-OT pA2 value = 7.19+/-0.06, peptide 4 is equipotent with Atosiban (pA2 = 7.05+/-0.05). However, with its significantly reduced anti-vasopressor potency, pA2 = approximately 5, it is approximately 18 times more selective for OT receptors with respect to VP V1a receptors than Atosiban. Since we have shown that V1a antagonism could be an unwanted side-effect in tocolytics, peptide 4 and some of the OT antagonists reported here have advantages over Atosiban and thus may be suitable candidates for evaluation as potential tocolytic agents for the treatment of preterm labour.     

Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases

Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.     

The role of the cross-link His-Tyr in the functional properties of the binuclear center in cytochrome c oxidase

Resonance Raman and Fourier transform infrared spectroscopies have been used to study the aa(3)-type cytochrome c oxidase and the Y280H mutant from Paracoccus denitrificans. The stability of the binuclear center in the absence of the Tyr(280)-His(276) cross-link is not compromised since heme a(3) retains the same proximal environment, spin, and coordination state as in the wild type enzyme in both the oxidized and reduced states. We observe two C-O modes in the Y280H mutant at 1966 and 1975 cm(-1). The 1975 cm(-1) mode is assigned to a gamma-form and represents a structure of the active site in which Cu(B) exerts a steric effect on the heme a(3)-bound CO. Therefore, the role of the cross-link is to fix Cu(B) in a certain configuration and distance from heme a(3), and not to allow histidine ligands to coordinate to Cu(B) rather than to heme a(3), rendering the enzyme inactive, as proposed recently (Das, T. K., Pecoraro, C., Tomson, F. L., Gennis, R. B., and Rousseau, D. L. (1998) Biochemistry 37, 14471-14476). The results provide solid evidence that in the Y280H mutant the catalytic site retains its active configuration that allows O(2) binding to heme a(3). Oxygenated intermediates are formed by mixing oxygen with the CO-bound mixed-valence wild type and Y280H enzymes with similar Soret maxima at 438 nm.     

Simultaneous resonance Raman detection of the heme a3-Fe-CO and CuB-CO species in CO-bound ba3-cytochrome c oxidase from Thermus thermophilus. Evidence for a charge transfer CuB-CO transition

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba3-cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the CuB-CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba3-oxidase. We assigned the bands at 507 and 568 cm(-1) to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm(-1) showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the alpha-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa3-oxidase, are discussed in terms of the proposed coupling of the alpha-and beta-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The CuB-CO complex was identified by its nu(CO) at 2053 cm(-1) and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent CuB(I) that is located at the O2 delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.     

Binding of O(2) and its reduction are both retarded by replacement of valine 279 by isoleucine in cytochrome c oxidase from Paracoccus denitrificans

The crystal structure of the heme-copper oxidases suggested a putative channel of oxygen entry into the heme-copper site of O(2) reduction. Changing a conserved valine near this center in cytochrome bo(3) of Escherichia coli to isoleucine caused a significant increase in the apparent K(M) for oxygen with little or no change in V(max), suggesting that oxygen diffusion had been partially blocked [Riistama, S., Puustinen, A., García-Horsman, A., Iwata, S., Michel, H., and Wikstr?m, M. (1996) Biochim. Biophys. Acta 1275, 1-4]. To study this phenotype further using rapid kinetic methods, the corresponding change (V279I) has been made in cytochrome aa(3) from Paracoccus denitrificans. In this mutant, the apparent K(M) for oxygen is 8 times higher than in the wild-type enzyme, whereas V(max) is decreased only to approximately half of the wild-type value. Flow-flash kinetic measurements show that the initial binding of oxygen to the heme of the binuclear site is indeed much slower in the mutant than in the wild-type enzyme. However, the subsequent phases of the reaction with O(2) are also slow although the pure heme-to-heme electron transfer process is essentially unperturbed. It is suggested that the mutation sterically hinders O(2) entry into the binuclear site and that it may also perturb the structure of local water molecules involved in proton transfer to this site.     

Demonstration by FTIR that the bo-type ubiquinol oxidase of Escherichia coli contains a heme-copper binuclear center similar to that in cytochrome c oxidase and that proper assembly of the binuclear center requires the cyoE gene product.

Amino acid sequence data have revealed that the bo-type ubiquinol oxidase from Escherichia coli is closely related to the eukaryotic aa3-type cytochrome c oxidases. In the cytochrome c oxidases, the reduction of oxygen to water occurs at a binuclear center comprised of heme a3 and Cu(B). In this paper, Fourier transform infrared (FTIR) spectroscopy of CO bound to the enzyme is used to directly demonstrate that the E. coli bo-type ubiquinol oxidase also contains a heme-copper binuclear center. Photolysis of CO ligated to heme o at low temperatures (e.g., 30 K) results in formation of a CO-Cu complex, showing that there is a heme-Cu(B) binuclear center similar to that formed by heme a3 and Cu(B) in the eukaryotic oxidase. It is further demonstrated that the cyoE gene product is required for the correct assembly of this binuclear center, although this polypeptide is not required as a component of the active enzyme in vitro. The cyoE gene product is homologous to COX10, a nuclear gene product from Saccharomyces cerevisiae, which is required for the assembly of yeast cytochrome c oxidase. Deletion of the cyoE gene results in an inactive quinol oxidase that is, however, assembled in the membrane. FTIR analysis of bound CO shows that Cu(B) is present in this mutant but that the heme-Cu(B) binuclear center is abnormal. Analysis of the heme content of the membrane suggests that the cyoE deletion results in the insertion of heme B (protoheme IX) in the binuclear center, rather than heme O.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the metabolism of eighteen-carbon 13C-unsaturated fatty acids in healthy women

Altered use of different dietary fatty acids may contribute to several chronic diseases, including obesity, noninsulin-dependent diabetes mellitus, and cardiovascular disease. However, few comparative data are available to support this link, so the goal of the present study was to compare the metabolism of [(13)C]oleate, [(13)C]alpha-linolenate, [(13)C]elaidate, and [(13)C]linoleate through oxidation and incorporation into plasma lipid fractions and adipose tissue. Each tracer was given as a single oral bolus to six healthy women. Samples were collected over 8 days, and (13)C was analyzed using isotope ratio mass spectrometry. At 9 h postdose, cumulative oxidation was similar for [(13)C]elaidate, [(13)C]oleate, and [(13)C]alpha-linolenate (19 +/- 1%, 20 +/- 4%, and 19 +/- 3% dose, respectively). Significantly lower oxidation of [(13)C]linoleate (12 +/- 4% dose; P < 0.05) was accompanied by its higher incorporation into plasma phospholipids and cholesteryl esters. Abdominal adipose tissue was enriched with [(13)C]alpha-linolenate, [(13)C]elaidate, or [(13)C]linoleate within 6 h. The percentage linoleate in plasma phospholipids correlated positively with [(13)C]linoleate and [(13)C]elaidate oxidation, indicating a potential role of background diet. Conversion of [(13)C]linoleate and [(13)C]alpha-linolenate to longer chain polyunsaturates was a quantitatively minor route of utilization.     

Differential coupling through Val-344 and Tyr-442 of trimethylamine dehydrogenase in electron transfer reactions with ferricenium ions and electron transferring flavoprotein

Modeling studies of the trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH-ETF) electron transfer complex have suggested potential roles for Val-344 and Tyr-442, found on the surface of TMADH, in electronic coupling between the 4Fe-4S center of TMADH and the FAD of ETF. The importance of these residues in electron transfer, both to ETF and to the artificial electron acceptor, ferricenium (Fc(+)), has been studied by site-directed mutagenesis and stopped-flow spectroscopy. Reduction of the 6-(S)-cysteinyl FMN in TMADH is not affected by mutation of either Tyr-442 or Val-344 to a variety of alternate side chains, although there are modest changes in the rate of internal electron transfer from the 6-(S)-cysteinyl FMN to the 4Fe-4S center. The kinetics of electron transfer from the 4Fe-4S center to Fc(+) are sensitive to mutations at position 344. The introduction of smaller side chains (Ala-344, Cys-344, and Gly-344) leads to enhanced rates of electron transfer, and likely reflects shortened electron transfer "pathways" from the 4Fe-4S center to Fc(+). The introduction of larger side chains (Ile-344 and Tyr-344) reduces substantially the rate of electron transfer to Fc(+). Electron transfer to ETF is not affected, to any large extent, by mutation of Val-344. In contrast, mutation of Tyr-442 to Phe, Leu, Cys, and Gly leads to major reductions in the rate of electron transfer to ETF, but not to Fc(+). The data indicate that electron transfer to Fc(+) is via the shortest pathway from the 4Fe-4S center of TMADH to the surface of the enzyme. Val-344 is located at the end of this pathway at the bottom of a small groove on the surface of TMADH, and Fc(+) can penetrate this groove to facilitate good electronic coupling with the 4Fe-4S center. With ETF as an electron acceptor, the observed rate of electron transfer is substantially reduced on mutation of Tyr-442, but not Val-344. We conclude that the flavin of ETF does not penetrate fully the groove on the surface of TMADH, and that electron transfer from the 4Fe-4S center to ETF may involve a longer pathway involving Tyr-442. Mutation of Tyr-442 likely disrupts electron transfer by perturbing the interaction geometry of TMADH and ETF in the productive electron transfer complex, leading to less efficient coupling between the redox centers.     

Probing molecular structure of dioxygen reduction site of bacterial quinol oxidases through ligand binding to the redox metal centers

Cytochromes bo and bd are structurally unrelated terminal ubiquinol oxidases in the aerobic respiratory chain of Escherichia coli. The high-spin heme o-CuB binuclear center serves as the dioxygen reduction site for cytochrome bo, and the heme b595-heme d binuclear center for cytochrome bd. CuB coordinates three histidine ligands and serves as a transient ligand binding site en route to high-spin heme o one-electron donor to the oxy intermediate, and a binding site for bridging ligands like cyanide. In addition, it can protect the dioxygen reduction site through binding of a peroxide ion in the resting state, and connects directly or indirectly Tyr288 and Glu286 to carry out redox-driven proton pumping in the catalytic cycle. Contrary, heme b595 of cytochrome bd participate a similar role to CuB in ligand binding and dioxygen reduction but cannot perform such versatile roles because of its rigid structure.