Phosphorescence has been observed at 1.2 K both for C60 dissolved in a decaline/cyclohexane glass and for single crystals of C60. The phosphorescence spectrum of C60 in the glass reveals a weak... Show morePhosphorescence has been observed at 1.2 K both for C60 dissolved in a decaline/cyclohexane glass and for single crystals of C60. The phosphorescence spectrum of C60 in the glass reveals a weak origin at 798.1 +/- 0.5 nm, while most of the intensity shows up in vibronic transitions. The phosphorescence of crystalline C60 consists of two parts. One of these, with the origin of its phosphorescence spectrum at 826 nm, derives probably from a shallow C60 X-trap although an excitonic origin cannot be excluded. The other, with the origin of its phosphorescence spectrum at 862 nm, originates from triplet states previously recognized by magnetic resonance experiments. These concern excitations delocalized over pairs of C60 molecules, so-called mini-excitons that are deep X-traps in the C60 crystal. Show less
Andrew, C.R.; Yeom, H.; Valentine, J.S.; Karlsson, B.G.; Pouderoyen, G. van; Canters, G.W.; ... ; Bonander, N. 1994
A series of metalloprotein mutants with novel copper cysteinate coordination environments has been probed by resonance Raman (RR) spectroscopy. These include H117G, M121E, and M121K mutants of... Show moreA series of metalloprotein mutants with novel copper cysteinate coordination environments has been probed by resonance Raman (RR) spectroscopy. These include H117G, M121E, and M121K mutants of Pseudomonas aeruginosa azurin and H46C, H80C, and H120C mutants of yeast CuZn-superoxide dismutase. In each case, excitation within a (Cys)S --> Cu charge transfer band leads to the enhancement of multiple vibrational modes of the copper cysteinate moiety. The predominant Cu-S stretching vibration, v(Cu-S), located in the 300-450 cm(-1) region, can be identified by (i) its large S- and Cu-isotope shifts, (ii) its high RR intensity, and (iii) its role as the generator of combination bands. The v(Cu-S) frequency appears to be a sensitive indicator of Cu-S(Cys) bond strength and, hence, copper coordination geometry. In the case of type 1 (T1) sites, the increased influence of the weak axial ligand upon moving from a trigonal planar (axial EPR) toward a more tetrahedral (rhombic EPR) geometry is associated with a decrease in v(Cu-S) from similar to 420 to similar to 350 cm(-1). In the case of type 2 (T2) sites with four strong ligands, v(Cu-S) undergoes further decreases from similar to 350 to similar to 310 cm(-1) as the geometry becomes more tetragonal. The Cu-S bond is successively weakened by trans ligand effects as the geometry approaches square planar. The decreased strength of the Cu-S(Cys) bond is further reflected in the increased strength of the adjacent S-C bond whose stretching frequency varies from similar to 750 cm(-1) for axial T1 sites to similar to 765 cm(-1) for T2 sites. The similar to 100-cm(-1) range in v(Cu-S) corresponds to a change in Cu-S(Cys) bond distance from similar to 2.13 Angstrom for an axial T1 site to similar to 2.29 Angstrom, for a tetragonal T2 site. The overlap of T1 and T2 v(Cu-S) frequencies near 350 cm(-1) shows that both types of Cu site can have similar Cu-S(Cys) bond strengths, despite their different EPR and optical characteristics, and points to a continuum of geometries linked through a tetrahedral structure which we describe as a T1.5 intermediate Cu site. Show less
The relation between quinone (QA) binding and electron transport in reaction centers (RCs) of photosynthetic purple bacteria is investigated, using electron spin polarization (ESP) X-band (9 GHz)... Show moreThe relation between quinone (QA) binding and electron transport in reaction centers (RCs) of photosynthetic purple bacteria is investigated, using electron spin polarization (ESP) X-band (9 GHz) EPR as a tool to probe for structural changes resulting from charge separation and stabilization and from replacing the native QA molecule with other quinones. We present a study of possible changes in Q(A)-binding that might be responsible for the remarkably prolonged Lifetime of the charge-separated state at cryogenic temperatures for RCs of Rhodobacter sphaeroides R26 cooled under illumination [Kleinfeld, D., et al. (1984) Biochemistry 23, 5780-5786]. It is shown that this effect is not caused by a major reorientation of the chromophores. Furthermore, we studied the effects of structurally different quinones functioning as primary electron acceptor in different purple bacteria. With simulations of ESP X-band spectra of the spin-polarized secondary radical pair P.(+)Q(A)(.-) in menaquinone-reconstituted, Zn2+-substituted RCs of Rb. sphaeroides R26, we show that quinone reconstitution is highly selective for site and orientation. Furthermore, we find that a very small exchange interaction between P-.+ and Q(A)(.-) (\J(PQ)\ similar to 1 mu T) is needed to account accurately for the observed relative line intensities at X-band, without affecting the accuracy of the simulations of reported ESP K-band spectra [Fuchsle, G., et al. (1993) Biochim. Biophys. Acta 1142, 23-35; Van der Est, A., et al. (1993) Chem. Phys. Lett. 212, 561-568]. This pronounced influence of small values for J(PQ) On the X-band ESP line shape results from cancellation effects of absorptive and emissive contributions to the spectrum, such that small shifts can be observed. The exchange interaction has opposite sign for the native, ubiquinone-containing RC [viz. J(P.UQ) (-0 8 +/- 0.2) mu T] and the menaquinone-substituted RC [J(P.MK) (+0.3 +/- 0.2)mu T]. The implications of these observations for electron-transport theory are discussed. Show less
Electron transfer reactions of the type 1 copper protein amicyanin from Thiobacillus versutus with [Co(terpy)(2)](2+) (terpy = 2,2':6',2''-terpyridine) as a reductant for AmCuII, and [Fe(CN)(6)](3-... Show moreElectron transfer reactions of the type 1 copper protein amicyanin from Thiobacillus versutus with [Co(terpy)(2)](2+) (terpy = 2,2':6',2''-terpyridine) as a reductant for AmCuII, and [Fe(CN)(6)](3-) and [Co(phen)(3)](3+) (phen = 1,10-phenanthroline) as oxidants for AmCul have been explored by stopped-flow kinetic studies at 25 degrees C, I = 0.100 M (NaCl). The reaction with [Co(terpy)(2)](2+) is a straightforward single-stage process, whereas both the oxidations give biphasic kinetics. The first stages of the latter exhibit an active-site 'switch-off' mechanism previously assigned as a protonation (and dissociation) of the His-96 ligand of AmCul. From the studies with [Co(phen)(3)](3+) an acid dissociation constant pK(a), of 6.6 and-rate constant at the higher pH of 7.1 x 10(3) M(-1) s(-1) are obtained. The reaction with [Fe(CN)(6)](3-) gives similar behaviour with a pk(a) of 6.6, and from the fitting procedure adopted, a rate constant at the higher pH of 8.3 x 10(6) M(-1) s(-1), which is at the upper limit of the stopped-flow range. The second stage of the reaction is explained by the formation of a less-reactive form of AmCul in amounts of between 5 and 40% depending on the pH. The rate law in the case of [Co(ph)(3)](3+) is independent of oxidant concentration, shows little independence on pH and gives rate constants (a) in the range 0.07-012 s(-1). For the more reactive [Fe(CN)(6)](3-) the rate equation is k(2abs) = a + b[Fe(CN)(6)(3-)], with a varying between 0.13 and 0.15 s(-1) and b = 680 M(-1) s(-1). The similar values of a for the two reactions are consistent with a common rate-controling intramolecular isomerisation step. The bimolecular rate constant b is approximate to 10(4) times smaller than the rate constant for the first stage of the [Fe(CN)(6)](3-) oxidation at pH approximate to 8. The nature of the structural differences between the two forms of AmCul is considered. Show less
Köhler, J.; Brouwer, A.C.J.; Groenen, E.J.J.; Schmidt, J. 1994
We report the observation of the broadening of a magnetic-resonance transition between triplet sublevels of a single pentacene molecule owing to the interaction of the triplet electron spin with a... Show moreWe report the observation of the broadening of a magnetic-resonance transition between triplet sublevels of a single pentacene molecule owing to the interaction of the triplet electron spin with a single C-13 nuclear spin. Analysis of this broadening allows the assignment of particular features in the fluorescence-excitation spectrum to pentacene molecules containing C-13 isotopes in specific positions. Show less
Ubbink, M.; Warmerdam, G.C.M.; Campos, A.P.; Teixeira, M.; Canters, G.W. 1994
By changing Met64 into a glutamate by means of site-directed mutagenesis a negative charge was introduced into the hydrophobic patch of azurin from Pseudomonas aeruginosa. The three-dimensional... Show moreBy changing Met64 into a glutamate by means of site-directed mutagenesis a negative charge was introduced into the hydrophobic patch of azurin from Pseudomonas aeruginosa. The three-dimensional structure of the protein and the structure of the metal site in particular, appear unaffected by the mutation. The observed change of the midpoint potential of the mutant of 28 mV is ascribed to the deprotonation of Glu64. The electron-self-exchange rate constant equals that of the wild-type protein at pH 4.5 but decreases by almost two orders of magnitude at high pH. Electron transfer is inhibited only when both of the reacting azurin molecules have an ionized glutamate at position 64 in their hydrophobic patch. Electron transfer at a graphite electrode is slowed down by the presence of the negative charge in the hydrophobic patch. The results demonstrate once again that the Cu-ligand His117 in the hydrophobic patch is the likely entry and exit point for electrons. This observation holds both for the (homogeneous) electron-self-exchange reaction in solution as well as for the (heterogenous) reaction at an electrode. Show less
Koorevaar, P.; Kes, P.H.; Koshelev, A.E.; Aarts, J. 1994
The electron spin polarization (ESP) of the triplet of the primary donor (P-3) in, reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis shows an anisotropic temperature... Show moreThe electron spin polarization (ESP) of the triplet of the primary donor (P-3) in, reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis shows an anisotropic temperature dependence (Van Wijk, F.G.H. and Schaafsma, T.J. (1988) Biochim. Biophys. Acta 936, 236). The reported inversion of the initial electron spin polarization (IESP) for the canonical Y-direction of P-3 at 100 K has been explained by means of coherent S-T-z mixing in the radical pair, due to a fast relaxing electron spin on the iron-quinone acceptor complex X (Here, P.J., Hunter, D.A., Van Wijk, F.G.H., Schaafsma, T.J. and Hoff, AJ. (1988) Biochim. Biophys. Acta 936, 249). Using direct-detection EPR, we show that at 100 K the IESP in randomly oriented samples is not inverted for the canonical Y-direction of P-3. Furthermore, in single crystals the IESP at 100 K is shown to be almost zero for the complete YZ-plane of P-3. Since X(.-) shows a strong g-anisotropy, the model of Here et al., in wg-anisotropy, the model of Here et al., in which polarization-inversion only occurs when the effective g-value of X(.-) is around g = 2.00, is inadequate to explain the temperature-dependent changes of the IESP. Therefore, we conclude that anisotropic fast spin-lattice relaxation in the radical pair triplet state is the origin of the temperature dependence of the ESP. The inversion for the canonical Y-direction under continuous illumination is the result of the interplay of spin-lattice relaxation in P-3 and its triplet decay rates, in combination with changes in the IESP. Show less
Field-swept electron spin echo spectroscopy at 95 GHz on a frozen solution and a single crystal of azurin from Pseudomonas aeruginosa has enabled the determination of the complete g-tensor. Highly... Show moreField-swept electron spin echo spectroscopy at 95 GHz on a frozen solution and a single crystal of azurin from Pseudomonas aeruginosa has enabled the determination of the complete g-tensor. Highly accurate principal values have been obtained (g(x) = 2.0393 +/- 0.0004, g(y) = 2.0568 +/- 0.0007, g(z) = 2.273 +/- 0.004), and the orientation of the principal axes of the g-tensor with respect to the copper site has been established. One of the principal axes makes an angle of 15 degrees with the Cu-S delta(Met121) bond. The other axes lie almost in the NNS plane and are rotated by 24 degrees with respect to the Cu-S gamma(Cys112) bond. The g-tensor does not corroborate descriptions of the copper site in terms of effective C-3v, C-2v, or C-s symmetry. The observed orientation of the principal axes presents a sensitive point of reference for future quantum-chemical considerations. Show less
Hoitink, C.W.G.; Driscoll, P.C.; Hill, H.A.O.; Canters, G.W. 1994
Complete sequential H-1 and N-15 resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) = 14 000, 129 residues) from Alcaligenes denitrificans have been obtained... Show moreComplete sequential H-1 and N-15 resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) = 14 000, 129 residues) from Alcaligenes denitrificans have been obtained at pH 5.5 and 32-degrees-C using homo- and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Comparison of the resonance assignments for the backbone protons with those of Pseudomonas aeruginosa azurin, which is 68% homologous in its amino acid sequence and has a very similar three-dimensional structure, showed a high similarity in chemical shift positions. After adjustment for random coil contributions the mean difference in N H chemical shifts is 0.00 ppm (root mean square width = 0.30 ppm), whereas for C(alpha) protons the mean difference is 0.09 ppm (root mean square width = 0.23 ppm). Characteristic NOE connectivities and 3J(HNalpha) values were used to determine the secondary structure of azurin in solution. Two beta-sheets, one helix, and nine tight and four helical turns were identified, and some long-range NOE contacts were found that connect the helix with the beta-sheets. The secondary structure obtained is in agreement with the structure derived from X-ray diffraction data [Baker, E. N. (1988) J. Mol. Biol. 203, 1071-1095]. Studies of the hydration of the protein in the vicinity of the copper ligand residue His117 revealed that the solvent-exposed N(epsilon2)H of His117 is in slow exchange with the bulk solvent. However, no evidence was obtained for the presence of a long-lived water molecule at the position corresponding to a well-defined water molecule observed in the crystal structures of A. denitrificans and Ps. aeruginosa azurin. Show less
