Vibrational modes of ubiquinone in cytochrome bo(3) from Escherichia coli identified by Fourier transform infrared difference spectroscopy and specific (13)C labeling.

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).     

Direct evidence for the protonation of aspartate-75, proposed to be at a quinol binding site, upon reduction of cytochrome bo3 from Escherichia coli

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 Wikstr?m, 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.     

Electrochemical and FTIR spectroscopic characterization of the cytochrome bc1 complex from Paracoccus denitrificans: evidence for protonation reactions coupled to quinone binding

The cytochrome bc(1) complex from Paracoccus denitrificans and soluble fragments of its cytochrome c(1) and Rieske ISP subunits are characterized by a combined approach of protein electrochemistry and FTIR difference spectroscopy. The FTIR difference spectra provide information about alterations in the protein upon redox reactions: signals from the polypeptide backbone, from the cofactors, and from amino acid side chains. We describe typical modes for conformational changes in the polypeptide and contributions of different secondary structure elements. Signals attributed to the different cofactors can be presented on the basis of selected potential steps. Modes associated with bound quinone are identified by comparison with spectra of quinone in solution at 1656, 1642, and 1610 cm(-1) and between 1494 and 1388 cm(-1), as well as at 1288 and 1262 cm(-1). Signals originating from the quinone bound at the Q(o) site can be distinguished. On the basis of the infrared data, the total quinone concentration is determined to be 2.6-3.3 quinones per monomer, depending on preparation conditions. The balance of evidence supports the double-occupancy model. Interestingly, the amplitude of the band at 1746 cm(-1) increases with quinone content, reflecting a protonation reaction of acidic groups. In this context, the involvement of glutamates and/or aspartates in the vicinity of the Q(o) site is discussed on the basis of recently determined crystal structures.     

Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.     

The effects of quinone analogues on cytochrome b6 reduction and oxidation in a reconstituted system

The reconstituted system containing Photosystem I, plastocyanin and the cytochrome b6-f complex is used to study the effects of various quinone analogues on the redox behavior of cytochrome b6. The effects of DBMIB, DNP-INT and HQNO are compared in an attempt to discern the modes of action of these quinone analogues. Both DBMIB and DNP-INT are potent inhibitors of the plastocyanin reductase activity of the isolated cytochrome complex. However, while DBMIB abolished the oxidant-induced reduction of cytochrome b6, DNP-INT only inhibited about 25% of the net reduction. On the other hand, HQNO does not show any significant inhibition of plastocyanin reductase activity of the isolated cytochrome complex at concentrations up to 20 microM. An enhancement of the net amount of cytochrome b6 reduced is observed in the presence of HQNO. Both DNP-INT and HQNO inhibited the dark oxidation rate of cytochrome b6. The possible identity of the oxidant for cytochrome b6 is discussed. Plastoquinone is concluded to be the most likely candidate. DNP-INT is concluded to have at least two sites of inhibition in the cytochrome complex. The implications of these findings on quinone functions in the cytochrome b6-f complex are discussed.     

FTIR spectroscopic characterization of the cytochrome aa3 from Acidianus ambivalens: evidence for the involvement of acidic residues in redox coupled proton translocation

The aa(3)-type quinol oxidase from Acidianus ambivalens is a divergent member of the heme-copper oxidases superfamily, namely, concerning the putative channels for intraprotein proton conduction. In this study, we used electrochemically induced FTIR difference spectroscopy to identify residues involved in redox-coupled protonation changes. In the spectral region characteristic for the nu(C=O) mode from protonated aspartic or glutamic acid side chains, a number of prominent features can be observed between 1790 and 1710 cm(-)(1), clearly indicating the reorganization or protonation of more than four protonatable residues upon electron transfer. A direct comparison of the Fourier-transform infrared difference spectra at different pH values reveals the noteworthy high pK of >8 for some of these residues, and the protonation of two of them. These acidic residues may play a role in the proton transport to the oxygen reducing site, in proton pumping pathways, or in protonation reactions concomitant with quinone reduction. Whereas the residues contributing between 1790 and 1750 cm(-)(1) have the typical position of an aspartic/glutamic acid side chain buried in the protein, a position closer to the surface is suggested for the residues contributing below approximately 1730 cm(-)(1). The possible involvement of residues contributing between 1750 and 1720 cm(-)(1) in the quinone binding site is demonstrated on the basis of experiments in the presence and absence of ubiquinone-2 and of the native electron carrier of the A. ambivalens respiratory chain, caldariella quinone. Most signals seen here are not observable in comparable spectra of typical members of the heme copper oxidase superfamily and thus reflect unique features of the enzyme from the hyperthermoacidophilic archaeon A. ambivalens.     

Study on the redox state dependent gamma(CH) vibrational modes of the c-type heme

Absorbance Fourier transform infrared (FTIR) spectra of model compounds for heme proteins such as protoporphyrin-IX, hemin, and hematin have been directly compared to the data of electrochemically induced FTIR difference spectra of small c-type proteins, i.e., microperoxidase-11, and cytochrome c. A band at 840-830 cm(-1) occurring in all studied samples dominated the spectra. The position of this vibrational mode depends on pH and the oxidation state, and could be assigned to the gamma(CH) mode of the porphyrin ring. Further features, such as the ring vibrations sensitive for the presence of iron and its oxidation state, are shown in the low-frequency infrared region between 750 and 650 cm(-1).     

The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli

Cytochrome bo₃ is the major respiratory oxidase located in the cytoplasmic membrane of Escherichia coli when grown under high oxygen tension. The enzyme catalyzes the 2-electron oxidation of ubiquinol-8 and the 4-electron reduction of dioxygen to water. When solubilized and isolated using dodecylmaltoside, the enzyme contains one equivalent of ubiquinone-8, bound at a high affinity site (Q_H). The quinone bound at the Q_H site can form a stable semiquinone, and the amino acid residues which hydrogen bond to the semiquinone have been identified. In the current work, it is shown that the tightly bound ubiquinone-8 at the Q_H site is not displaced by ubiquinol-1 even during enzyme turnover. Furthermore, the presence of high affinity inhibitors, HQNO and aurachin C1–10, does not displace ubiquinone-8 from the Q_H site. The data clearly support the existence of a second binding site for ubiquinone, the Q_L site, which can rapidly exchange with the substrate pool. HQNO is shown to bind to a single site on the enzyme and to prevent formation of the stable ubisemiquinone, though without displacing the bound quinone. Inhibition of the steady state kinetics of the enzyme indicates that aurachin C1–10 may compete for binding with quinol at the Q_L site while, at the same time, preventing formation of the ubisemiquinone at the Q_H site. It is suggested that the two quinone binding sites may be adjacent to each other or partially overlap.     

Characterization of mutations in crucial residues around the Q(o) binding site of the cytochrome bc complex from Paracoccus denitrificans

The protonation state of residues around the Q(o) binding site of the cytochrome bc(1) complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site-directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme b(L) edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild-type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Q(o) site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P.denitrificans as well. The protonated residue E295 is proposed to change the hydrogen-bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X-ray structures.     

Modification of the phylloquinone in the A1 binding site in photosystem I studied using time-resolved FTIR difference spectroscopy and density functional theory

A phylloquinone molecule (2-methyl-3-phytyl-1,4-naphthoquinone) occupies the A1 binding site in photosystem I. Previously, we have obtained A1(-)/A1 FTIR difference spectra using labeled and unlabeled photosystem I particles and proposed assignments for many of the bands in the spectra [Sivakumar, V., Wang, R., and Hastings, G. (2005) Biochemistry 44, 1880-1893]. In particular, we suggested that a negative/positive band at 1654/1495 cm(-1) in A1(-)/A1 FTIR DS is due to a C=O/C-:O mode of the neutral/anionic phylloquinone, respectively. To test this hypothesis, we have obtained A1(-)/A1 FTIR DS for menG mutant PS I particles. In menG mutant PS I, phylloquinone in the A1 binding site is replaced with an analogue in which the methyl group at position 2 of the quinone ring is replaced with a hydrogen atom (2-phytyl-1,4-naphthoquinone). In A1(-)/A1 FTIR DS obtained using menG mutant PS I particles, we find that the 1654/1495 cm(-1) bands are upshifted by approximately 6 cm(-1). To test if such upshifts are likely for C=O/C-:O modes of neutral/anionic phylloquinone, we have used density functional theory to calculate the "anion minus neutral" infrared difference spectra for both phylloquinone and its methyl-less analogue. We have also undertaken calculations in which the C4=O carbonyl group of phylloquinone and its methyl-less analogue are hydrogen bonded (to a water or leucine molecule). We find that, irrespective of the hydrogen bonding state of the C4=O group, the C=O/C-:O modes of neutral/reduced phylloquinone are indeed expected to be upshifted by at least 6 cm(-1) upon replacement of the methyl group at position 2 with hydrogen. The calculations also suggest that certain C=C/C-:C modes of neutral/reduced phylloquinone do not shift upon replacement of the methyl group. On the basis of these calculated results, we suggest which bands in the A1(-)/A1 FTIR DS may be associated with C=C/C-:C modes of neutral/reduced phylloquinone, respectively.     

Vibrational spectroscopy to study the properties of redox-active tyrosines in photosystem II and other proteins

Tyrosine radicals play catalytic roles in essential metalloenzymes. Their properties--midpoint potential, stability...--or environment varies considerably from one enzyme to the other. To understand the origin of these properties, the redox tyrosines are studied by a number of spectroscopic techniques, including Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopy. An increasing number of vibrational data are reported for the (modified-) redox active tyrosines in ribonucleotide reductases, photosystem II, heme catalase and peroxidases, galactose and glyoxal oxidases, and cytochrome oxidase. The spectral markers for the tyrosinyl radicals have been recorded on models of (substituted) phenoxyl radicals, free or coordinated to metals. We review these vibrational data and present the correlations existing between the vibrational modes of the radicals and their properties and interactions formed with their environment: we present that the nu7a(C-O) mode of the radical, observed both by RR and FTIR spectroscopy at 1480-1515 cm(-1), is a sensitive marker of the hydrogen bonding status of (substituted)-phenoxyl and Tyr*, while the nu8a(C-C) mode may probe coordination of the Tyr* to a metal. For photosystem II, the information obtained by light-induced FTIR difference spectroscopy for the two redox tyrosines TyrD and TyrZ and their hydrogen bonding partners is discussed in comparison with those obtained by other spectroscopic methods.     

Binding of HQNO to beef-heart sub-mitochondrial particles

1. The fluorescence spectra of HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) in water at pH 7.5 show an emission maximum at 480 nm and an excitation maximum at 355 nm.2. The fluorescence is enhanced by binding to bovine serum albumin, and is completely quenched by binding to sub-mitochondrial particles of beef heart.3. Binding experiments reveal specific binding of HQNO to sub-mitochondrial particles with a dissociation constant of 64 nM and, depending on the protein concentration, a considerable amount of aspecific binding.4. The concentration of specific binding sites for HQNO is identical with that of antimycin-binding sites. Furthermore, the presence of antimycin prevents the binding of HQNO and antimycin releases HQNO from its binding site.5. The binding of HQNO is not sensitive to the redox state of the respiratorychain components.6. Inhibition of electron transfer by HQNO is caused by binding to the specific binding site.7. The relation between inhibition of NADH or succinate oxidation and saturation of the binding site is hyperbolic.8. The increase in the reduction level of cytochrome b on addition of HQNO in the presence of succinate and oxygen, either in the presence or absence of cyanide, does not parallel the inhibition of overall electron transfer.9. All data can be quantitatively described and analysed using the model for electron transfer proposed by Wikström and Berden in 1972 (Wikström, M. K. F. and Berden, J. A. (1972) Biochim. Biophys. Acta 283, 403–420).     

Electrochemically induced FTIR difference spectroscopy in the mid- to far infrared (200 microm) domain: a new setup for the analysis of metal-ligand interactions in redox proteins

We report the setup of an electrochemical cell with chemical-vapor deposition diamond windows and the use of a Bruker 66 SX FTIR spectrometer equipped with DTGS and Si-bolometer detectors and KBr and mylar beam splitters, to record on the same sample, FTIR difference spectra corresponding to the structural changes associated with the change in redox state of active sites in proteins in the whole 1800-50 cm(-1) region. With cytochrome c we show that reliable reduced-minus-oxidized FTIR difference spectra are obtained, which correspond to single molecular vibrations. Redox-sensitive IR modes of the cytochrome c are detected until 140 cm(-1) with a good signal to noise. This new setup is promising to analyze the infrared spectral region where metal-ligand vibrations are expected to contribute and to extend the analysis of vibrational properties to metal sites or redox states not accessible to (resonance) Raman spectroscopy.     

Direct and mediated electron transfer between intact succinate:quinone oxidoreductase from Bacillus subtilis and a surface modified gold electrode reveals redox state-dependent conformational changes

Succinate:quinone oxidoreductase (SQR) from Bacillus subtilis consists of two hydrophilic protein subunits comprising succinate dehydrogenase, and a di-heme membrane anchor protein harboring two putative quinone binding sites, Q(p) and Q(d). In this work we have used spectroelectrochemistry to study the electronic communication between purified SQR and a surface modified gold capillary electrode. In the presence of two soluble quinone mediators the midpoint potentials of both hemes were revealed essentially as previously determined by conventional redox titration (heme b(H), E(m)=+65 mV, heme b(L), E(m)=-95 mV). In the absence of mediators the enzyme still communicated with the electrode, albeit with a reproducible hysteresis, resulting in the reduction of both hemes occurring approximately at the midpoint potential of heme b(L), and with a pronounced delay of reoxidation. When the specific inhibitor 2-n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Q(d) in B. subtilis SQR, was added together with the two quinone mediators, rapid reductive titration was still possible which can be envisioned as an electron transfer occurring via the HQNO insensitive Q(p) site. In contrast, the subsequent oxidative titration was severely hampered in the presence of HQNO, in fact it completely resembled the unmediated reaction. If mediators communicate with Q(p) or Q(d), either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Q(p) depended on the redox state of the hemes. When both hemes were reduced, and Q(d) was blocked by HQNO, quinone-mediated communication via the Q(p) site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain.     

Redox infrared markers of the heme and axial ligands in microperoxidase: bases for the analysis of c-type cytochromes

Structural changes accompanying the change in the redox state of microperoxidase-8 (MP8), the heme-octapeptide obtained from cytochrome c, and its complexes with (methyl)imidazole ligands were studied by electrochemically induced Fourier transform IR (FTIR) difference spectroscopy. To correlate with confidence IR modes with a specific electronic state of the iron, we used UV-vis and electron paramagnetic resonance spectroscopy to define precisely the heme spin state in the samples at the millimolar concentration of MP8 required for FTIR difference spectroscopy. We identified four intense redox-sensitive IR heme markers, nu38 at 1,569 cm(-1) (ox)/1,554 cm(-1) (red), nu42 at 1,264 cm(-1) (ox)/1,242 cm(-1) (red), nu43 at 1,146 cm(-1) (ox), and nu44 at 1,124-1,128 cm(-1) (ox). The intensity of nu42 and nu43 was clearly enhanced for low-spin imidazole-MP8 complexes, while that of nu44 increased for high-spin MP8. These modes can thus be used as IR markers of the iron spin state in MP8 and related c-type cytochromes. Moreover, one redox-sensitive band at 1,044 cm(-1) (red) is attributed to an IR marker specific of c-type hemes, possibly the delta(CbH3)(2,4) heme mode. Other redox-sensitive IR bands were assigned to the MP8 peptide backbone and to the fifth and sixth axial heme ligands. The distinct IR frequencies for imidazole (1,075 cm(-1)) and histidine (1,105 cm(-1)) side chains in the imidazole-MP8 complex allowed us to provide the first direct determination of their pKa at pH 9 and 12, respectively.     

Three functionally different cytochrome b redox centres in pigeon heart mitochondria.

Three functionally different cytochrome b redox centres, apparently of high metabolic activity, were detected in intact pigeon heart mitochondria; cytochrome b(1), b(m) and b(h), with maxima of absorption at 556.6 (State 5), 560.6, and 564.5 nm, respectively (alpha-bands, 77K). 2. Cytochrome (b(l) was reduced in the presence of either antimycin or HQNO (2-heptyl-4-hydroxyquinoline N-oxide). The absorption maximum was shifted by dithionite, cyanide, NNN'N'-tetramethyl-p-phenylenediamine + ascorbate, HQNO and antimycin. The spectra obtained on simultaneous or successive addition of HQNO and antimycin favoured the assumption of a common binding site for the two inhibitors. 3. Cytochrome b(m) was reduced in the presence of HQNO, but not in the presence of antimycin. No shifts of absorption maximum was observed. 4. Cytochrome b(h) was reduced in the presence of antimycin. HQNO was unable to cause reduction of this cytochrome by endogenous substrates. The absorption maximum was shifted to lower wavelength by organic solvents. It was inseparable from that of cytochrome b(m) in the presence of 0.4% ethanol. 5. The pattern of reduction in the presence of HQNO or antimycin demonstrates the functional difference of the three redox centres and appears incompatible wih a linear respiratory chain.     

Ligand, cofactor, and residue vibrations in the catalytic site of endothelial nitric oxide synthase

A study of bovine endothelial nitric oxide synthase by Fourier transform infrared (FTIR) spectroscopy in the 1000-2500 cm(-)(1) range is reported. Binding of CO to the reduced enzyme gives two heme(II)-CO nu(C)(-)(O) stretches (1927 and 1904 cm(-)(1)) which appear to be in rapid equilibrium. Photolysis of this heme(II)-CO compound is accompanied by perturbation of the local fine structure around the catalytic site giving vibrational changes of protein backbone, substrate, amino acid residues, and cofactors, to which heme, substrate arginine, and catalytic site residues contribute. Possible assignments of vibrations to heme, substrate arginine, and catalytic site residues are discussed. The discussion of assignments is informed by known structures, absorbance frequencies, and extinction coefficients of residues and cofactors, analysis of H(2)O-D(2)O exchange effects, analysis of substrate (14)N-(15)N (guanidinium)-arginine exchange effects, and comparison with the nNOS isoform (which differs in the replacement of asparagine 368 with an aspartate within the substrate binding site). The FTIR data can be modeled on the known structure of the catalytic site and indicate the extent of modulation of vibrational modes upon photolysis of the CO compound.     

Kinetic evidence for involvement of two cytochrome b-563 hemes in photosynthetic electron transport

The effect of 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) on the kinetics of cytochrome b-563 and cytochrome c² turnovers following single-turnover flashes was measured in isolated heterocysts. Low concentrations of HQNO (below 3 μM) blocked reoxidation of cytochrome b-563, whereas higher concentrations (above 5 μM) resulted in additional inhibition of cytochrome b-563 oxidation and also inhibited reduction of cytochrome b-563 and cytochrome c. Similar effects on cytochrome b-563 reduction and reoxidation were obtained with a combination of 5 μM HQNO and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (1–7 μM). In HQNO-inhibited heterocysts, cytochrome c reduction following a flash occurred in three phases with half-times of 0.5, 2.8 and 45 ms. The second phase nearly equalled the cytochrome b-563 reduction in half-time and magnitude. In the presence of HQNO, the reoxidation of cytochrome b-563 following two closely spaced actinic flashes displayed biphasic kinetics. The two phases correspond to reoxidation of cytochrome b-563 in which one or both of the cytochrome b-563 hemes in the cytochrome b–f complex are reduced. These results are interpreted in terms of a Q-loop in which HQNO, at low concentrations, blocks the site of rapid cytochrome b-563 reoxidation and at higher concentrations, also inhibits the site of electron donation by plastoquinol to the cytochrome b-f complex.     

Complex formation of cytochrome P450cam with Putidaredoxin. Evidence for protein-specific interactions involving the proximal thiolate ligand.

We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam). These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.     

FTIR difference spectroscopy in combination with isotope labeling for identification of the carbonyl modes of P700 and P700+ in photosystem I

Room temperature, light induced (P700(+)-P700) Fourier transform infrared (FTIR) difference spectra have been obtained using photosystem I (PS I) particles from Synechocystis sp. PCC 6803 that are unlabeled, uniformly (2)H labeled, and uniformly (15)N labeled. Spectra were also obtained for PS I particles that had been extensively washed and incubated in D(2)O. Previously, we have found that extensive washing and incubation of PS I samples in D(2)O does not alter the (P700(+)-P700) FTIR difference spectrum, even with approximately 50% proton exchange. This indicates that the P700 binding site is inaccessible to solvent water. Upon uniform (2)H labeling of PS I, however, the (P700(+)-P700) FTIR difference spectra are considerably altered. From spectra obtained using PS I particles grown in D(2)O and H(2)O, a ((1)H-(2)H) isotope edited double difference spectrum was constructed, and it is shown that all difference bands associated with ester/keto carbonyl modes of the chlorophylls of P700 and P700(+) downshift 4-5/1-3 cm(-1) upon (2)H labeling, respectively. It is also shown that the ester and keto carbonyl modes of the chlorophylls of P700 need not be heterogeneously distributed in frequency. Finally, we find no evidence for the presence of a cysteine mode in our difference spectra. The spectrum obtained using (2)H labeled PS I particles indicates that a negative difference band at 1698 cm(-1) is associated with at least two species. The observed (15)N and (2)H induced band shifts strongly support the idea that the two species are the 13(1) keto carbonyl modes of both chlorophylls of P700. We also show that a negative difference band at approximately 1639 cm(-1) is somewhat modified in intensity, but unaltered in frequency, upon (2)H labeling. This indicates that this band is not associated with a strongly hydrogen bonded keto carbonyl mode of one of the chlorophylls of P700.