Conformational dependence of electron transfer across de novo designed metalloproteins.

Flash photolysis and pulse radiolysis measurements demonstrate a conformational dependence of electron transfer rates across a 16-mer helical bundle (three-helix metalloprotein) modified with a capping CoIII(bipyridine)3 electron acceptor at the N terminus and a 1-ethyl-1'-ethyl-4,4'- bipyridinium donor at the C terminus. For the CoIII(peptide)3-1-ethyl-1'-ethyl-4,4'-bipyridinium maquettes, the observed transfer is a first order, intramolecular process, independent of peptide concentration or laser pulse energy. In the presence of 6 M urea, the random coil bundle (approximately 0% helicity) has an observed electron transfer rate constant of kobs = 900 +/- 100 s-1. In the presence of 25% trifluoroethanol (TFE), the helicity of the peptide is 80% and the kobs increases to 2000 +/- 200 s-1. Moreover, the increase in the rate constant in TFE is consistent with the observed decrease in donor-acceptor distance in this solvent. Such bifunctional systems provide a class of molecules for testing the effects of conformation on electron transfer in proteins and peptides.

Noncovalent binding of heme induces a compact apocytochrome c structure.

Mitochondrial holocytochrome c contains heme that is covalently attached to the protein in a reaction catalyzed by the enzyme cytochrome c heme lyase. In the absence of heme, apocytochrome c, the precursor to holocytochrome c, is unfolded. We find that purified apocytochrome c binds noncovalently to heme. Binding is accompanied by changes in the optical absorption spectrum of heme and by quenching of the tryptophan fluorescence of the protein. The affinity of apocytochrome c for heme, as well as the stoichiometry of binding, appears to depend on whether or not cyanide is present and on the oxidation state of heme. Under reducing conditions, in the presence of cyanide, the association appears to be 1:1, with a binding constant of about 10(7) M-1. Under oxidized conditions, there may be multiple hemes bound per molecule of apocytochrome c. Upon binding to heme, apocytochrome c exhibits a mobility similar to that of holocytochrome c in gel filtration chromatography and velocity gradient ultracentrifugation, indicating that the heme-protein complex adopts a structure that is almost as compact as that of holocytochrome c. Changes in the circular dichroism spectrum of apocytochrome c are consistent with an increase in the alpha-helical content of the protein on binding heme. The compact structure of the noncovalent heme-apocytochrome c complex may represent an intermediate in the de novo folding of cytochrome c.

Effects of surface amino acid replacements in cytochrome c peroxidase on intracomplex electron transfer from cytochrome c.

Transient absorption techniques were used to measure the intracomplex electron transfer rates between four recombinant yeast cytochrome c peroxidases and iso-1 cytochrome c (cytc). The binding affinities and catalytic activities with cytc were previously examined [Corin et al. (1991) Biochemistry 30, 11585]. The four include a wild-type peroxidase (ECcP) and three others, each of which has one surface aspartic acid converted to lysine at position 37, 79, or 217. These sites have been suggested to be within or proximal to the recognition site for cytc. These mutants conduct electron transfer with cytc but differ with respect to the ionic strength profiles of their limiting rate constants. At pH and mu = 114 mM, ECcP and D217K show similar limiting rate constants for electron transfer with cytc, k(lim), of ca. 2000 s-1. In the same peroxidase concentration range, the D37K mutant exhibits a k(obs) of ca. 100 s-1. Instability of the compound I form of D79K prevented a complete study of the intracomplex kinetics of this mutant by this technique. At pH 6 and low ionic strength (8 mM), D37K exhibits a dramatic increase in k(obs) to ca. 800 s-1 while the other two recombinants show a marked decrease to values < 150 s-1. D37K displays much lower affinity for cytc than do the other peroxidases at higher ionic strengths [Hake et al. (1992) J. Am. Chem. Soc. 114, 5442], thus preventing adequate complexation necessary for efficient electron transfer. Variations in binding affinity do not explain the more subtle ionic strength kinetic profile observed for D217K.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of surface amino acid replacements in cytochrome c peroxidase on complex formation with cytochrome c.

Site-directed mutagenesis was employed to examine the role played by specific surface residues in the activity of cytochrome c peroxidase. The double charge, aspartic acid to lysine, point mutations were constructed at positions 37, 79, and 217 on the surface of cytochrome c peroxidase, sites purported to be within or proximal to the recognition site for cytochrome c in an electron-transfer productive complex formed by the two proteins. The resulting mutant peroxidases were examined for catalytic activity by steady-state measurements and binding affinity by two methods, fluorescence binding titration and cytochrome c affinity chromatography. The cloned peroxidases exhibit similar UV-visible spectra to the wild-type yeast protein, indicating that there are no major structural differences between the cloned peroxidases and the wild-type enzyme. The aspartic acid to lysine mutations at positions 79 and 217 exhibited similar turnover numbers and binding affinities to that seen for the "wild type-like" cloned peroxidase. The same change at position 37 caused more than a 10-fold decrease in both turnover of and binding affinity for cytochrome c. This empirical finding localizes a primary recognition region critical to the dynamic complex. Models from the literature proposing structures for the complex between peroxidase and cytochrome c are discussed in light of these findings.

Protein conformational "annealing": binding to cytochrome c refolds the active site region of recombinant cytochrome c peroxidase.

When initially isolated with heme reconstitution, recombinant cytochrome c peroxidase molecules exhibit a conformation, revealed by visible spectra which observably differ from the corresponding holo proteins isolated from yeast. Binding yeast iso-1 cytochrome c to these recombinant cytochrome c peroxidases (either in solution or via an affinity column) catalyses a local refolding of the recombinant proteins to a form that is indistinguishable from the native (yeast) protein.

Photo-induced electron ejection from the reduced copper of Pseudomonas aeruginosa azurin.

The reduced form of Pseudomonas aeruginosa azurin exhibits an enhanced absorbance in the UV compared to that of the oxidized protein. This enhancement has also been observed for azurins from other bacterial species and for another type I copper protein, plastocyanin. Pulsed laser excitation of the reduced azurin in the region of enhanced absorbance at 308 nm results in single photon, rapid (less than 30 ns) oxidation of the copper center and formation of the hydrated electron with a quantum yield of 0.05. The hydrated electron reacts in the expected manner with scavengers such as nitrous oxide, oxygen, acetone and nitromethane. In the absence of scavengers, the electron reacts with the protein, including the disulfide bond, to form the disulfide radical anion, observed at 410 nm. The overall photophysical event involves a charge-transfer to solvent transition although the existence of intermediate states can not be excluded.

Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy.

The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission intensities and anisotropies of eosin and erythrosin immobilized in poly(methyl methacrylate). The fluorescence data are compared with those from phosphorescence emission measurements and with theoretical predictions. Triplet-state lifetimes were obtained in 5 mM phosphate buffer, pH 7.0, of concanavalin A labeled with eosin, tetramethylrhodamine, and fluorescein and of alpha 2-macroglobulin labeled with the first two probes. In the case of labeled concanavalin A, iodide quenching measurements gave bimolecular rate constants of approximately 10(9) M-1 s-1. The usefulness of FRS for studying protein-cell interactions is exemplified with eosin-labeled concanavalin A bound to living A-431 human epidermoid carcinoma cells. Finally, the advantages and disadvantages of the technique are compared to those of the alternative phosphorescence emission method.

Proflavin binding to poly[d(A-T)] and poly[d(A-br5U)]: triplet state and temperature-jump kinetics.

The delayed fluorescence properties of proflavin have been exploited in studies of the excited-state binding kinetics of the dye to poly[d(A-T)] and its brominated analogue poly[d(A-br5U)] at room temperature and pH 7. The two analyzed luminescence decay times of the DNA-dye complex are dependent on the total nucleic acid concentration. This dependence is shown to reflect a temporal coupling of the intrinsic delayed emission decay rates with the dynamic chemical kinetic binding processes in the excited state. Temperature-jump kinetic studies conducted on the brominated polymer and corresponding information on poly[d(A-T)] from a previous study [Ramstein, J., Ehrenberg, M., & Rigler, R. (1980) Biochemistry 19, 3938-3948] provide complementary information about the ground state. In the ground state, the poly[d(A-T)]-proflavin complex has one chemical relaxation time, which reaches a plateau at high DNA concentrations. The brominated DNA-dye complex exhibits two relaxation times: a faster relaxation mode that behaves similarly to that for the unhalogenated DNA and a slower relaxation mode that is apparent at high DNA concentrations. The ground-state kinetic data are analyzed in terms of two alternative models incorporating series and parallel reaction schemes. The former consists of two sequential binding steps--a fast bimolecular process followed by a monomolecular step--while the latter consists of two coupled bimolecular steps. A similar analysis for the excited-state data yields reasonable kinetic constants only for the series model, which, in accordance with previous proposals for acridine intercalators, consists of a fast outside binding step followed by intercalation of the dye. A comparison of the ground- and excited-state kinetic parameters reveals that the external binding process is much stronger and the intercalation is much weaker in the excited state. That the excited-state data are only consistent with the series model suggests that delayed luminescence studies may provide a general tool for distinguishing between the two kinetic mechanisms. In particular, we demonstrate the use of delayed luminescence spectroscopy as a tool for probing dynamic DNA-ligand interactions in solution.

The microsecond rotational motions of eosin-labelled fatty acids in multilamellar vesicles.

The rotational properties of two eosin-labelled fatty acids of different alkyl chain length have been studied in large multilamellar dimyristoylphosphatidylcholine vesicles. The location of the probes at the surface region were ascertained by quenching experiments using a hydrophilic divalent cation solubilized in the aqueous phase (Cu2+) and a hydrophobic aromatic aniline (N,N-dimethylaniline) associated with the lipid. Phosphorescence anisotropy measurements reveal that above the phospholipid phase transition the polarization of eosin luminescence decays monoexponentially in the micro-to-millisecond time range, while below the phase transition a biexponential decay is observed. A model is proposed which attributes the time constants to two separate motions, discrete jumps or 'flipping' of the eosin moiety within restricted boundaries and long-axis rotation. The value of the time-independent term changes with probe position and temperature and reflects orientational constraints imposed by lipid-chromophore interactions. The implications of these results for the study of protein rotations in membranes are discussed.

The size of the lactose permease derived from rotational diffusion measurements.

The lactose permease of Escherichia coli was labeled with eosinyl-maleimide, reconstituted into vesicles of dimyristoylphosphatidylcholine and subjected to time-dependent phosphorescence anisotropy measurements in order to determine the rotational diffusion coefficient. By comparison with bacteriorhodopsin, the diffusion coefficient is evaluated in terms of an effective radius of the lactose permease in the plane of the membrane. This radius amounts to 20 +/- 2 A which implies that the lactose permease is a monomer. The monomeric state is maintained in the presence of a membrane potential.

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.