Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c.

Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.     

Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching?

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.     

ATP induces a conformational change in lipid-bound cytochrome c

Resonance energy transfer studies using a pyrene-labeled phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphoglycerol (donor) and the heme (acceptor) of cytochrome c (cyt c) have indicated that ATP causes changes in the conformation of the lipid-bound protein (Ryt?maa, M., Mustonen, P., and Kinnunen, P. K. J. (1992) J. Biol. Chem. 267, 22243-22248). Accordingly, after binding cyt c via its so called C-site to neat phosphatidylglycerol liposomes (mole fraction of PG = 1.0) has commenced, further quenching of donor fluorescence is caused by ATP, saturating at 2 mm nucleotide. ATP-induced conformational changes in liposome-associated cyt c could be directly demonstrated by CD in the Soret band region (380-460 nm). The latter data were further supported by time-resolved spectroscopy using the fluorescent cyt c analog with a Zn(2+)-substituted heme moiety. A high affinity ATP-binding site has been demonstrated in cyt c (Craig, D. B., and Wallace, C. J. A. (1993) Protein Sci. 2, 966-976) that is compromised by replacing the invariant Arg(91) to norleucine. Although no major effects on conformation and function of cyt c were concluded due to the modification, a significantly reduced effect by ATP on the lipid-bound [Nle(91)]cyt c was evident, implying that this modulation is mediated via the Arg(91)-containing binding site.     

Cytochrome c and cytochrome c peroxidase complex as studied by resonance Raman spectroscopy.

Complex formation between ferricytochrome c peroxidase (CCP) and ferricytochrome c from yeast [cyt(Y)] and horse heart [cyt(H)] was studied by resonance Raman spectroscopy. On the basis of a detailed spectral analysis of the free proteins, it was possible to attribute changes in the spectra of the complexes to the individual proteins. At pH 7.0 both cyt(Y) and cyt(H) binding induces an increase in the six-coordinate low-spin configuration of CCP from 9% to 19% at the expense of the five-coordinate high-spin state, which drops from 84% to 74%. In the free and complexed state, CCP exhibits a constant fraction of the six-coordinate high-spin form (approximately 7%). In addition to affecting the coordination state, there is also a cyt-specific structural response of CCP to complexation. In the cyt(Y)-CCP complex, the peripheral vinyl and propionate substituents of CCP are more rigidly fixed in the protein matrix, whereas binding of cyt(H) only slightly perturbs the conformations of these side chains. The biological significance of the conformational changes in CCP are discussed. In contrast to CCP, there are no detectable structural changes in either cyt(Y) or cyt(H) upon complex formation.     

Cytochrome c interaction with yeast cytochrome b2. Heme distances determined by energy transfer in fluorescence resonance

Fluorescent derivatives of cytochrome c were prepared by replacing the heme iron with closed-shell metals such as zinc or tin. The iron-free derivatives of cytochrome c bind to yeast lactate dehydrogenase (cytochrome b2) stoichiometrically and with high affinity. Spectral overlap exists between the fluorescence of porphyrin, Zn(II) or Sn(IV) cytochrome c and the absorption of the heme of cytochrome b; therefore dipole-dipole interaction is possible as predicted by F?rster's theory of energy transfer. Changes in the fluorescence yield and the fluorescent decay profile of the cytochrome c derivatives are consistent with the view that the heme distance is sufficiently close for dipolar interactions. The distance calculated from the data depends upon assumptions in the theory for energy transfer and uncertainties in the experiment. It can be argued that due to the symmetry of the metalloporphyrins the relative orientations of the two hemes do not introduce a significant uncertainty in the calculation. However the decay profiles of the iron-free cytochromes are complex, possibly reflecting structural rearrangement of the polypeptide chain during the fluorescent lifetime. The steady-state fluorescent yields would indicate that the mean distance is around 1.8 nm.     

Intrinsic tryptophan phosphorescence as a marker of conformation and oxygen diffusion in purified cytochrome oxidase.

Cytochrome oxidase exhibits phosphorescence from tryptophan in aqueous solution in the absence of oxygen. The lifetime for the resting reduced enzyme suspended in Tween-20 is around 30 ms at pH 8. The lifetime is longest between pH 7 and 8 and decreases with lowering of pH. Oxygen quenches the phosphorescence with a Stern-Volmer quenching constant of approximately 5 x 10(7) M-1.s-1 at 5 degrees C whereas cytochrome c has no effect. We interpret these results to indicate that room temperature tryptophan phosphorescence arises from tryptophan(s) in structured region(s) remote from the hemes and that the protein does not impose a significant barrier for the diffusion of oxygen.     

Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase.

Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.     

Interaction of electron acceptors with the excited triplet state of Zn cytochrome c

The decay rate of the excited triplet state of Zn cytochrome c was enhanced by electron acceptors including methyl viologen and ferric complexes of cyanide, oxalate, EDTA and cytochrome c at room temperature. Ferrous compounds were several orders of magnitude less effective than the respective ferric form in quenching the phosphorescence. In the presence of ferricytochrome c and ferricyanide the semilogarithmic plots of the decay curve showed an anomalous decay profile in which the rate of interaction appeared to accelerate after excitation. One explanation is that the quenching process was accelerated by a conformational change of the polypeptide chain around the excited triplet state porphyrin. Another explanation is that quenching occurs via an intermediate.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with Ki values of 4.5, 91, 200 and 330 microM, respectively. 2. The inhibition constant Ki of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme. 3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k1 of 33 M-1 . s-1 and a dissociation constant Kd of 3.9 mM. 4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M-1 . s-1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes. 5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome alpha 3-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

Reversible, nonionic, and pH-dependent association of cytochrome c with cardiolipin-phosphatidylcholine liposomes.

Membrane association of cytochrome c (cyt c) was monitored by the efficiency of resonance energy transfer from a pyrene-fatty acid containing phospholipid derivative (1-palmitoyl-2[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphocholine (PPHPC)) to the heme of cyt c. Liposomes consisted of 85 mol% egg phosphatidylcholine (egg PC), 10 mol% cardiolipin, and 5 mol% PPHPC. Cardiolipin was necessary for the membrane binding of cyt c over the pH range studied, from 4 to 7. In accordance with the electrostatic nature of the membrane association of cyt c at neutral pH both 2 mM MgCl2 and 80 mM NaCl dissociated cyt c from the vesicles completely. At neutral pH also adenine nucleotides in millimolar concentrations were able to displace cyt c from liposomes, their efficiency decreasing in the sequence ATP > ADP > AMP. In addition, both CTP and GTP were equally effective as ATP. The detachment of cyt c from liposomes by nucleotides is likely to result from a competition between cardiolipin and the nucleotides for a common binding site in cyt c. When pH was decreased to 4 there was a small yet significant increase in the apparent affinity of cyt c to cardiolipin containing liposomes. Notably, at pH 4 the above nucleotides as well as NaCl and MgCl2 were no longer able to dissociate cyt c and, on the contrary, they slightly enhanced the quenching of pyrene fluorescence by cyt c. The above results do suggest that the membrane association of cyt c at acidic pH was non-ionic and presumably due to hydrogen bonding. The pH-dependent binding of cyt c to membranes was fully reversible. Accordingly, in the presence of sufficient concentrations of either nucleotides or salts rapid detachment and membrane association of cyt c could be induced by varying pH between neutral and acidic values, respectively.     

The binding of porphyrin cytochrome c to yeast cytochrome c peroxidase. A fluorescence study of the number of sites and their sensitivity to salt

Porphyrin c, the iron-free derivative of cytochrome c, is a reasonably good model for cytochrome c binding to cytochrome c peroxidase (CcP). It binds with the same stoichiometry but only one-quarter as tightly as cytochrome c. CcP (resting, FeIII) and CcP X CN can both bind up to two molecules of porphyrin c. The binding of the first porphyrin c is tight (kd = 1 X 10(-9) M, pH 6, ionic strength mu = 0, 4 degrees C) and results in quenching of the porphyrin c fluorescence. The binding is sensitive to ionic strength. The binding of the second porphyrin c is looser (Kd unknown) and does not result in quenching of the porphyrin fluorescence. The binding of porphyrin c to the cyano form and the resting forms of CcP cannot be distinguished by our methods. ES is the first acceptor of electrons from c(II) and can bind at least two molecules of porphyrin c. The binding of the first porphyrin c is extremely tight, results in substantial quenching and is insensitive to ionic strength. The binding of porphyrin c to the loose site (Kd = 2 X 10(-9) M, pH 6, 4 degrees C, mu = 0) results, unlike the resting and cyano forms, in quenching of fluorescence of the second porphyrin c. The binding of the second porphyrin c to ES is sensitive to ionic strength. The calculated distances between porphyrin c and the hemes of CcP(FeIII) and ES are approximately 2.5 nm.     

Electron paramagnetic resonance of the excited triplet state of metal-free and metal-substituted cytochrome c.

The photoactivated metastable triplate states of the porphyrin (free-base, i.e., metal-free) zinc and tin derivatives of horse cytochrome c were investigated using electron paramagnetic resonance. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range 3.8-150 K are discussed in terms of porphyrin-protein interactions. The zero-field splitting parameters D for the free-base, Zn and Sn derivatives are 465 x 10(-4), 342 x 10(-4) and 353 x 10(-4) cm-1, respectively, and are temperature invariant over the temperature ranges studied. AN E value at 4 K of 73 x 10(-4) cm-1 was obtained for Zn cytochrome c, larger than any previously found for Zn porphyrins derivatives of hemeproteins, showing that the heme site of cytochrome c imposes an asymmetric field. Though the E value for Zn cytochrome c is large, the geometry of the site appears quite constrained, as indicated by a spectral line shape showing a single species. Intersystem crossing occurred predominantly to the T2 > zero-field spin sublevel. EPR line shape changes with respect to temperature of Zn cyt c are interpreted in terms of vibronic coupling, and a maximum Jahn-Teller crystal-field splitting of approximately 180 cm-1 is obtained. Sn cytochrome c in comparison with the Zn protein exhibits a photoactivated triplet line shape that is less well resolved in the X-Y region. The magnitude of E value is approximately 60 x 10(-4) cm-1 at 4 K; its value rapidly tends toward zero with increasing temperature, from which a value for the Jahn-Teller crystal-field splitting of > or = 40 cm-1 is estimated. In contrast to those for the metal cytochromes, the magnitude of E value for the free-base derivative was essentially zero at all temperatures studied. This finding is discussed as a consequence of an excited-state tautomerization process that occurs even at 4 K.     

Cytochrome c interaction with membranes. Absorption and emission spectra and binding characteristics of iron-free cytochrome c.

A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidence indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics on Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in either the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absence of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heme to heme distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state.     

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.     

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.     

Binding of benzo[a]pyrene by purified cytochrome P-450c

Benzo[a]pyrene (BP) fluorescence-emission intensities in phospholipid micelles are quantitatively described over a broad range of lipid and BP concentrations by excitation that is linearly dependent upon BP concentration and an offsetting excimer quenching that is dependent upon the square of the BP concentration. The fluorescence of BP is quenched by the presence of cytochrome P-450c in proportion to the concentration of the cytochrome in the micelles and in accord with stoichiometric complex formation. Parallel optical titrations indicate a change in spin state of P-450c to a predominantly high-spin state that correlates directly with the percentage fluorescence quenching of complexed BP. Neither change occurs with five other purified forms of rat liver P-450 that have low activity in BP metabolism. N-Octylamine, a ligand that binds to the heme of P-450, competitively inhibits both the spin-state changes and the fluorescence quenching in equal proportion. The Kd for the interaction of BP with P-450c is exceptionally low (10 nM) relative to the Km for monooxygenation (ca. 1 microM). Decreasing the concentration of either dilauroylphosphatidylcholine or dioleoylphosphatidylcholine concomitantly increases the high-spin state (from 30% to 80%) of fully complexed P-450c and the fluorescence quenching (50-100%) of the complexed BP (half-maximal at 80 micrograms of lipid/mL). It is concluded that spin state and fluorescence quenching both reflect the same changes in the interaction of the BP with the P-450 heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein--protein docking of electron transfer complexes: cytochrome c oxidase and cytochrome c

Electron transferring protein complexes form only transiently and the crystal structures of electron transfer protein--protein complexes involving cytochrome c could so far be determined only for the pairs of yeast cytochrome c peroxidase (CcP) with iso-1-cytochrome c (iso-1-cyt c) and with horse heart cytochrome c (cyt c). This article presents models from computational docking for complexes of cytochrome c oxidase (COX) from Paracoccus denitrificans with horse heart cytochrome c, and with its physiological counterpart cytochrome c552 (c552). Initial docking is performed with the FTDOCK program, which permits an exhaustive search of translational and rotational space. A filtering procedure is then applied to reduce the number of complexes to a manageable number. In a final step of structural and energetic refinement, the complexes are optimized by rigid-body energy minimization with the molecular mechanics package CHARMM. This methodology was first tested on the CcP:iso-1-cyt c complex, in which the complex with the lowest CHARMM energy has an RMSD from the crystal structure of only 1.8 A (C(alpha) carbon atoms). Notably, the crystal conformation has an even lower energy. The same procedure was then applied to COX:cyt c and COX:c552. The lowest-energy COX:cyt c complex is very similar to a docking model previously described for the complex of bovine cytochrome c oxidase with horse heart cytochrome c. For the COX:c552 complex, cytochrome c552 is found in two different orientations, depending on whether it is docked against COX from a two-subunit or from a four-subunit crystal structure, respectively. Both conformations are discussed critically in the light of the available experimental data.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of zinc-substituted cytochrome c

Zinc-substituted cytochrome c has been widely used in studies of protein-protein interactions and photo-induced electron transfer reactions between proteins. However, the coordination geometry of zinc in zinc-substituted cyt c has not yet been determined; two different opinions about the coordination have been reached. Here the solution structures of zinc-substituted cytochrome c that might be five-coordinated and six-coordinated have been refined separately by using (1)H NMR spectroscopy, and the zinc coordination geometry was determined just by NOE distance constraints. Structural analysis of the energy-minimized average solution structures of both the pentacoordinated and hexacoordinated geometries indicate that that zinc in zinc-substituted cyt c should be bound to both His18 and Met80, which means that the zinc is six-coordinated. RMSD values of the family of 25 six-coordinated structures from the average structure are 0.66+/-0.13 A and 1.09+/-0.16 A for the backbone and all heavy atoms, respectively. A statistical analysis of the structure indicates its satisfactory quality. Comparison of the solution structure of the six-coordinated energy-minimized average structure of zinc-substituted cytochrome c with the solution structure of reduced cytochrome c reveals that for the overall folding the secondary structure elements are very close. The availability of the structure provides for a better understanding of the protein-protein complex and for electron transfer processes between Zn cyt c and other metalloproteins.