pH dependence of the reduction-oxidation reaction of azurin with cytochrome c-551: role of histidine-35 of azurin in electron transfer.

A fluorescence quenching experiment confirms that in the redox reaction between cytochrome c-551 and azurin, protein complexing is negligible. Azurin-pH indicator T-jump experiments show that Pseudomonas aeruginosa (Ps.) azurin exhibits a slow time constant, tau, in its return to pH equilibrium but Alcaligenes faecalis (Alc.) azurin does not. The decrease of l/tau with increasing pH shows that the rate-determining process is a slow transformation of the imidazolium form of histidine-35 from a conformation where it cannot ionize to one in which it can. The fast relaxation time constant of the redox reaction varies little with pH, but the slow time constant increased by a factor of approximately 2.5 increasing pH between pH 5 and pH 8. The corresponding amplitudes, especially the slow one, vary with pH. On the basis of all the present evidence it is concluded that, while some differences of redox reactivity do occur on protonation, these differences are not major. In general, the two proteins cyt c-551 and azurin react with each other with rates only weakly dependent upon pH. A classical pH titration was carried out on the reduced and oxidized form of Ps. and Alc. azurin with the result that two protons were released between pH 6 and pH 8, in the former from His-35 and -83 and in the latter from His-83 and Ala-1.

Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics.

On the basis of redox kinetic studies, Rosen and Pecht [Rosen, P. & Pecht, I. (1976) Biochemistry 15, 775-786] postulated a slowly attained (approximately equal to 0.1 sec) conformational equilibrium between two forms of reduced azurin from the bacterium Pseudomonas aeruginosa, one form being faster in electron transfer. NMR investigations have shown that at pH 7 there are two forms of reduced azurin exchanging slowly with each other, differing in the presence or absence of a proton on the imidazole side chain of histidine-35. Rosen et al. [Rosen, P., Segal, M. & Pecht, I. (1981) Eur. J. Biochem. 120, 339-344] observed that the azurin from the bacterium Alcaligenes faecalis shows no such slowly attained equilibrium between two forms. Therefore, a 1H NMR study was carried out on this azurin with emphasis on the downfield region. A resonance was found at 7.95 ppm downfield that does not move with pH, is not seen in the oxidized protein, has the same pseudocontact shift in the Co(II) derivative as the C-2 proton of histidine-35 has in the Co(II) derivative of P. aeruginosa azurin, and, in the apoprotein, exhibits a typical protonation shift downfield at pH less than 5. Therefore, this resonance is assigned to the C-2 proton of histidine-35. The crystal structure of P. aeruginosa azurin shows that at pH 7 the imidazole side chain of histidine-35 is in a crevice within the protein, where its ring is adjacent and parallel to that of histidine-47, a copper ligand. The preceding observations combined with others show that the kinetics of some redox reactions involving azurin depend on the position of histidine-35. The implication is that there is a pathway for electron transport to the copper atom involving passage through histidine-35.

Resolution of the two metal binding sites of human serum transferrin by low-temperature excitation of bound europium (III).

Derivatives of apotransferrin have been prepared in which europium replaces iron at either one or both of the two metal ion binding sites. At low temperature (77 K), pH 7.0, two sharp absorption lines are seen by means of laser-induced fluorescence of the bound europium. The one at 579.88 nm (17 245 cm-1) is assigned to the C-terminal region A site, and the other at 579.26 nm (17 263 cm-1) is assigned to the N-terminal region B site. The lifetimes of the excited 5D0 states are 210 +/- 20 and 310 +/- 30 mus for the A and B sites, respectively. The energy difference between the two peaks is a function of pH, with the splitting decreasing from 0.62 nm (18.5 cm-1) at pH 7.0 to 0.15 nm (4.5 cm-1) at pH 8.0. This spectroscopic unequivalence may be explained by a charge difference of the liganding groups at sites A and B.

Distance between metal-binding sites in transferrin: energy transfer from bound terbium (III) to iron (III) or manganese (III).

The distance between the two metal-binding sites of human serum transferrin has been studied by observing energy transfer between an excited terbium ion bound at one site and a ferric (or manganic) ion bound at the other site of the same transferrin molecule. From the observed reduction in terbium lifetime (relative to that of terbium transferrin), it is concluded that the intersite distance is 3.55 +/ 0.45 nm. This distance is reconciled with two conflicting earlier reports that the separation between sites is greater than 4.3 nm [Luk, C.K. (1971) Biochemistry 10,2838-2844] or is equal to 2.5 +/ 0.2 nm [Meares, C.F., & Ledbetter, J.E. (1977) Biochemistry 16, 5178-5180]. The difficulty of accurately measuring the quantum yield of protein-bound terbium provides the principal source of uncertainty in these measurements.

Location of the heme groups in cytochrome cd1 oxidase from Pseudomonas aeruginosa.

The disposition of the heme groups in cytochrome cd1 oxidase from Pseudomonas aeruginosa is studied by emission spectroscopy. This protein of molecular weight 120 000 is composed of two monomers each with a heme c and a heme d1. It has been shown by electron microscopy to be oblong in shape and by preliminary X-ray crystallography to have a twofold axis of rotation. Three electronic energy donors, a singlet tryptophan, a triplet tryptophan, and an attached 8-dimethylamino-1-naphthalenesulfonyl group, all exhibit normal decay lifetimes. It follows that there are parts of the protein at least 80 A from the nearest heme. The conclusion is that the hemes are all at one end of the molecule.

Energy transfer among the chromophores in phycocyanins measured by picosecond kinetics.

Energy-transfer processes in the algal light-harvesting proteins, the phycocyanins, have been studied by means of picosecond absorption spectroscopy. After excitation at 530 nm, the absorption at several wavelengths in the range 480--669 nm decayed with a short time constant (picosecond) and a long time constant (greater than 1 ns). For C-phycocyanin, energy transfer from the beta to the alpha subunits is interpreted as being a likely candidate for the short time constant; the long time constant probably is the excitation lifetime of the chromophore on the alpha subunits. The time constants for energy transfer in monomers, trimers, and hexamers of C-phycocyanin extracted from a blue-green alga, Phormidium luridum, were measured as approximately 85, approximately 56, and approximately 32 ps, respectively. The corresponding time constant in the cryptomonad phycocyanin 645 from Chroomonas species was found to be less than 5 ps.

1H NMR and ESR studies of oxidized cytochrome c551 from Pseudomonas aeruginosa.

Near neutral pH, Fe(III) cytochrome c551 exhibits an ESR absorption due primarily to a single species with g values of 3.24, 2.06, and 1.48. These g values are somewhat different from those of horse heart cytochrome c and can be interpreted by the generalizations of Brautigan et al. [(1977) J. Biol. Chem. 252, 574] to be due to Fe binding by the imidazole anion of histidine rather than by neutral imidazole. The NMR spectrum of Fe(III) cytochrome c551 exhibits a number of hyperfine-shifted peaks whose pattern shows similarities to but many differences from that of horse heart cytochrome c. Variation in shifts of some of the peaks in the pH range 5--9 is ascribed to ionization of a somewhat buried propionic acid side chain (pK = 5.8) and to ionization of the N-terminal NH3+ group (pK = 7.7). At alkaline pH greater than 9.4, as shown by a variety of optical and ESR spectral changes, the Met-61 S ligand is replaced by other ligands.

Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens.

A strain of Pseudomonas fluorescens contains an azurin with no tryptophan and two tyrosines. This protein is interesting because it allows one to study both the structure of azurin and the emission of tyrosines in proteins. Comprehensive measurements were carried out including spectrophotometric and fluorimetric titration, fluorescence quantum yield, fluorescence polarization, and I- quenching. In the copper-containing protein, almost independent of the copper ion oxidation, the fluorescence quantum yield is approximately 60% of that of the apoprotein. The latter has the remarkable property that its quantum yield is even greater than free tyrosine. The two tyrosines in the metalloprotein have different pKa's, 10.75 and 12.78, but there is only one average pKa, 10.9 in the apoprotein. The polarization of the fluorescence at 310 nm (290-nm excitation) is 0.32 for the metalloproteins and 0.34 for the apoprotein. I- hardly quenches the fluorescence. The conclusion is that the two tyrosines are inaccesible to the solvent, located in nonpolar environments, larger than or equal to 20 A apart, and not adjacent to the disulfide bridge.

Study of the triplet state properties of tyrosines and tryptophan in azuring using optically detected magnetic resonance.

Optically detected magnetic resonance (ODMR) signals and phosphorescence spectra were seen of tyrosine in the P. aeruginosa and tryptophanless P. fluorescens azurins and of tryptophan in the former. This confirmed a conclusion from other experiments that the tryptophan of P. aeruginosa cannot effectively quench the singlet energy of both tyrosines. The ODMR signals were all very narrow, additional evidence that the chromophores are buried in the interior of the protein. Accurate values of the zero-field coupling constants D and E lead to a tentative correlation of D values with the red shift of the 0 leads to 0 peak of the phosphorescence spectrum. The environment of tryptophan in P. aeruginosa is the most hydrocarbon like of any tryptophan so far observed. The experiments raise a number of unanswered questions concerning rate processes. The intensities of the 2E transition of tyrosine and the phosphorescence of both tyrosine and tryptophan are substantially reduced when the copper is oxidized. Nevertheless the phsphorescence lifetimes are unaffected. A hole cannot be burned in the ODMR resonances. The homogeneously broadened lines may conceivably be a result of low-temperature proton tunnelling.

Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy.

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.

Time-resolved spectroscopy of hemoglobin and its complexes with subpicosecond optical pulses.

Subpicosecond optical pulses have been used to study the photolysis of hemoglobin conplexes. Photodissociation of carboxyhemoglobin is found to occur in less than 0.5 picosecond. In hemoglobin and oxyhemoglobin a nondissociative excited state recovery in 2.5 picoseconds is observed.

A proposed fluorimetric determination of unsaturated iron-binding capacity.

A completely new method for the analysis of uncombined iron-binding capacity (UIBC) in human serum is proposed. The method is based on the discovery by C. K. Luk that the fluorescence of terbium ion (Tb-3+) is enhanced enormously when it binds to an empty Fe-3+ binding site of transferrin. Tb-3+ cannot displace Fe-3+ from an occupied binding site on transferrin. The analytic procedure is to dilute the serum (in order to lower its ultraviolet absorbance), add a solution of Tb-3+, excite with ultraviolet radiation, and measure the intensity of the resulting green fluorescence. With proper calibration this intensity is a measure of UIBC. Analyses of 65 serum samples previously measured at a hospital laboratory show no obvious difficulty with the method. Its absolute accuracy has not been probed in detail.

Interaction of transition metal ions with ribonuclease A. II. The selective effects of Mn2+, Zn2+, Cd2+ and Hg2+ on the histidine magnetic resonance.

Zn2+, Cd2+ and Hg2+ inhibit ribonuclease but Mn2+ does not except at very high concentrations. By high resolution NMR one can detect in the pH range 5-8 the C-2 protons of histidines 105, 12, and 119. The inhibiting ions produce large shifts of the resonance of His-12 but not of His-105. On the other hand Mn2+ broadens the C-2 proton of His-105 much more than it does those of His-12 and 119. The selective shifts suggest that the mechanism of inhibition is binding at or near the active site of which His-12 and 119 are a part. The selective broadening is a consequence of binding of the Mn2+ to a site very far from the active site but closer to His-105.