Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation.

The particulate methane monooxygenase (pMMO) is the first enzyme in the C1 metabolic pathway in methanotrophic bacteria. As this enzyme converts methane into methanol efficiently near room temperature, it has become the paradigm for developing an understanding of this difficult C1 chemistry. pMMO is a membrane-bound protein with three subunits (PmoB, PmoA, and PmoC) and 12-14 coppers distributed among different sites. X-ray crystal structures that have revealed only three mononuclear coppers at three sites have neither disclosed the location of the active site nor the catalytic mechanism of the enzyme. Here we report a cyro-EM structure of holo-pMMO from Methylococcus capsulatus (Bath) at 2.5 Å, and develop quantitative electrostatic-potential profiling to scrutinize the nonprotein densities for signatures of the copper cofactors. Our results confirm a mononuclear CuI at the A site, resolve two CuIs at the B site, and uncover additional CuI clusters at the PmoA/PmoC interface within the membrane (D site) and in the water-exposed C-terminal subdomain of the PmoB (E clusters). These findings complete the minimal set of copper factors required for catalytic turnover of pMMO, offering a glimpse of the catalytic machinery for methane oxidation according to the chemical principles underlying the mechanism proposed earlier.

Excited-state backbone twisting of polyfluorene as detected from photothermal after-effects.

By means of time-resolved photoluminescence and photothermal techniques, after-effects from excited-state dynamics, energy migration, and conformational rearrangement of poly(9,9-di-n-octyl-2,7-fluorene) (PFO) and its homologues has been examined and interpreted with rotational potential maps from quantum mechanical calculations. Steady-state photoluminescence spectral changes and time-resolved photoluminescence measurements of oligofluorenes and PFO diluted in toluene suggest excited state ring torsion occurring within 30 ps of photoexitation. With all effects from internal conversion/intersystem crossing processes properly accounted for, we show that the conformational changes associated with this twisting motion can be quantitatively probed by means of photothermal methods. Results suggest mean torsion between neighboring fluorene units by ca. 40 degrees upon excitation, in agreement with the shift of rotational potential minimum from +/-40 degrees (and +/-140 degrees) in the ground state to +/-20 degrees (and +/-160 degrees) in the first excited singlet state according to results of quantum mechanical calculations.

The role of a beta-bulge in the folding of the beta-hairpin structure in ubiquitin.

It is known that the peptide corresponding to the N-terminal beta-hairpin of ubiquitin, U(1-17), can populate the monomeric beta-hairpin conformation in aqueous solution. In this study, we show that the Gly-10 that forms the bulge of the beta-turn in this hairpin is very important to the stability of the hairpin. The deletion of this residue to desG10(1-16) unfolds the structure of the peptide in water. Even under denaturing conditions, this bulge appears to be important in maintaining the residual structure of ubiquitin, which involves tertiary interactions within the sequence 1 to 34 in the denatured state. We surmise that this residual structure functions as one of the nucleation centers in the folding process and is important in stabilizing the transition state. In accordance with this idea, deleting Gly-10 slows down the refolding and unfolding rate by about one half.

Effects of turn residues in directing the formation of the beta-sheet and in the stability of the beta-sheet.

The designed peptide (denoted 20-mer, sequence VFITS(D)PGKTYTEV(D)PGOKILQ) has been shown to form a three-strand antiparallel beta-sheet. It is generally believed that the (D)Pro-Gly segment has the propensity to adopt a type II' beta-turn, thereby promoting the formation of this beta-sheet. Here, we replaced (D)Pro-Gly with Asp-Gly, which should favor a type I' turn, to examine the influence of different type of turns on the stability of the beta-sheet. Contrary to our expectation, the mutant peptide, denoted P6D, forms a five-residue type I turn plus a beta-bulge between the first two strands due to a one amino-acid frameshift in the hydrogen bonding network and side-chain inversion of the first beta-strand. In contrast, the same kind of substitution at (D)Pro-14 in the double mutant, denoted P6DP14D, does not yield the same effect. These observations suggest that the SDGK sequence disfavors the type I' conformation while the VDGO sequence favors a type I' turn, and that the frameshift in the first strand provides a way for the peptide to accommodate a disfavored turn sequence by protruding a bulge in the formation of the beta-hairpin. Thus, different types of turns can affect the stability of a beta-structure.

Structures and proton-pumping strategies of mitochondrial respiratory enzymes.

Enzymes of the mitochondrial respiratory chain serve as proton pumps, using the energy made available from electron transfer reactions to transport protons across the inner mitochondrial membrane and create an electrochemical gradient used for the production of ATP. The ATP synthase enzyme is reversible and can also serve as a proton pump by coupling ATP hydrolysis to proton translocation. Each of the respiratory enzymes uses a different strategy for performing proton pumping. In this work, the strategies are described and the structural bases for the action of these proteins are discussed in light of recent crystal structures of several respiratory enzymes. The mechanisms and efficiency of proton translocation are also analyzed in terms of the thermodynamics of the substrate transformations catalyzed by these enzymes.

ESEEM studies of succinate:ubiquinone reductase from Paracoccus denitrificans.

Electron spin-echo envelope modulation (ESEEM) spectroscopy has been performed in order to obtain structural information about the environment of the reduced [2Fe-2S] cluster (S-1 center), the oxidized [3Fe-4S] cluster (S-3 center), and the flavin semiquinone radical in purified succinate:ubiquinone reductase from Paracoccus denitrificans. Spectral simulations of the ESEEM data from the reduced [2Fe-2S] yielded nuclear quadrupole interaction parameters that are indicative of peptide nitrogens. We also observed a weak interaction between the oxidized [3Fe-4S] cluster and a peptide 14N. There was no evidence for coordination of any of the Fe atoms to 14N atoms of imidazole rings. The ESEEM data from the flavin semiquinone radical were more complicated. Here, evidence was obtained for interactions between the unpaired electron and only the two nitrogen atoms in the flavin ring.

Reaction of Escherichia coli cytochrome bo(3) and mitochondrial cytochrome bc(1) with a photoreleasable decylubiquinol.

In order to probe the reaction chemistry of respiratory quinol-oxidizing enzymes on a rapid time scale, a photoreleasable quinol substrate was synthesized by coupling decylubiquinol with the water-soluble protecting group 3',5'-bis(carboxymethoxy)benzoin (BCMB) through a carbonate linkage. The resulting compound, DQ-BCMB, was highly soluble in aqueous detergent solution, and showed no reactivity with quinol-oxidizing enzymes prior to photolysis. Upon photolysis in acetonitrile, 5, 7-bis(carboxymethoxy)-2-phenylbenzofuran, carbon dioxide, and decylubiquinol were formed. In aqueous media, free 3', 5'-bis(carboxymethoxy)benzoin was also produced. Photolysis of DQ-BCMB with a 308 nm excimer laser led to the release of the BCMB group in less than 10(-6) s. Decylubiquinol was released in the form of a carbonate monoester, which decarboxylated with an observed first-order rate constant of 195-990 s(-1), depending on the reaction medium. Yields of decylubiquinol as high as 35 microM per laser pulse were attained readily. In the presence of Escherichia coli cytochrome bo(3), photolysis of DQ-BCMB led to the oxidation of quinol by the enzyme with a rate that was limited by the rate of the decylubiquinol release. Mitochondrial cytochrome bc(1) reacted with photoreleased decylubiquinol with distinct kinetic phases corresponding to rapid b heme reduction and somewhat slower c heme reduction. Oxidation of photoreleased ubiquinol by this enzyme showed saturation kinetics with a K(m) of 3.6 microM and a k(cat) of 210 s(-1). The saturation behavior was a result of decylubiquinol being released as a carbonate monoester during the photolysis of DQ-BCMB and interacting with cytochrome bc(1) before decarboxylation of this intermediate yielded free decylubiquinol. The reaction of cytochrome bc(1) and photoreleased decylubiquinol in the presence of antimycin A led to monophasic b heme reduction, but also yielded slower quinol oxidation kinetics. The discrimination of kinetic phases in the reaction of cytochrome bc(1) with ubiquinol substrates has provided a means of exploring the bifurcation of electron transfer that is central to the operation of the Q-cycle in this enzyme.

Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal residues. This broad specificity was confirmed by the C-terminal ladder sequencing of several synthetic and natural peptides, including porcine N-acetyl-renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser.

Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers.

Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal alpha-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/PC ratios and the amounts of free peptides. Based on the above observations, possible mechanisms of the bilayer lysis induced by CB1 and CB3 on liposomes of different composition are discussed.

The effect of Glu75 of staphylococcal nuclease on enzyme activity, protein stability and protein unfolding.

Staphylococcal nuclease mutants, E57G and E75G, were generated. A comparison of the kinetic parameters both for mutants and wild-type protein shows that the Michaelis constants (Km) were almost identical for the wild-type protein and E57G mutant. An approximately 30-fold decrease in Km compared with the wild-type protein was observed for the E75G mutant. The turnover numbers for the enzyme (kcat) were higher with both the wild-type protein and the E57G mutant (3.88 +/- 0.21 x 103 s-1 and 3.71 +/- 0.28 x 103 s-1) than with the E75G mutant (3.04 +/- 0.02 x 102 s-1). The results of thermal denaturation with differential scanning microcalorimetry indicate that the excess calorimetric enthalpy of denaturations, DeltaHcal, was almost identical for the wild-type protein and E57G mutant (84.1 +/- 6.2 kcal.mol-1 and 79.3 +/- 7.1 kcal.mol-1, respectively). An approximately twofold decrease in DeltaHcal compared with the wild-type protein was observed for the E75G mutant (42.7 +/- 5.5 kcal.mol-1). These outcomes imply that Glu at position 75 plays a significant role in maintaining enzyme activity and protein stability. Further study of the unfolding of the wild-type protein and E75G mutant was conducted by using time-resolved fluorescence with a picosecond laser pulse. Two fluorescent lifetimes were found in the subnanosecond time range. The faster lifetime (tau2) did not generally vary with either pH or the concentration of guanidinium hydrochloride (GdmHCl) in the wild-type protein and the E75G mutant. The slow lifetime (tau1), however, did vary with these parameters and was faster as the protein is unfolded by either pH or GdmHCl denaturation. The midpoints of the transition for tau1 are pH 3.5 and 5.8 for the wild-type protein and E75G mutant, respectively, and the GdmHCl concentrations are 1.1 m and 0.6 m for the wild-type protein and E75G mutant, respectively. Parallel steady-state fluorescence measurements have also been carried out and the results are in general agreement with the time-resolved fluorescence experiments, indicating that Glu at position 75 plays an important role in protein unfolding.

Hydrogen exchange kinetics of proteins in denaturants: a generalized two-process model.

The recent progress in measurements on the amide hydrogen exchange (HX) in proteins under varying denaturing conditions, both at equilibrium and in transient relaxation, necessitates the development of a unifying theory which quantitatively relates the HX rates to the conformational energetics of the proteins. We present here a comprehensive kinetic model for the site-specific HX of proteins under varying solvent denaturing conditions based on the two-state protein folding model. The generalized two-process model considers both conformational fluctuations and residual protections, respectively, within the folded and unfolded states of a protein, as well as a global kinetic folding-unfolding transition between the two states. The global transition can be either rapid or slow, depending on the solvent condition for the protein. This novel model is applicable to the traditional equilibrium HX measurements in both EX2 and EX1 regimes, and also the recently introduced transient pulse-labeling HX experiments. A set of simple analytical equations is provided for quantitative interpretation of experimental data. The model emphasizes the use of full time-course of bi-exponential HX kinetics, rather than fitting time-course data to single rate constants, to obtain quantitative information about fluctuating conformers within the folded and unfolded states of proteins. This HX kinetic model naturally unfolds into a simple two-state and two-stage kinetic interpretation for protein folding. It suggests that the various observed intermediates of a protein can be interpreted as dominant isomers of either the folded or the unfolded state under different solvent conditions. This simple, minimalist's view of protein folding is consistent with various recent experimental observations on folding kinetics by HX.

Kinetic steps for alpha-helix formation.

The kinetics of alpha-helix formation in polyalanine and polyglycine eicosamers (20-mers) were examined using torsional-coordinate molecular dynamics (MD). Of one hundred fifty-five MD experiments on extended (Ala)20 carried out for 0.5 ns each, 129 (83%) formed a persistent alpha-helix. In contrast, the extended state of (Gly)20 only formed a right-handed alpha-helix in two of the 20 MD experiments (10%), and these helices were not as long or as persistent as those of polyalanine. These simulations show helix formation to be a competition between the rates of (a) forming local hydrogen bonds (i.e. hydrogen bonds between any residue i and its i + 2, i + 3, i + 4, or i + 5th neighbor) and (b) forming nonlocal hydrogen bonds (HBs) between residues widely separated in sequence. Local HBs grow rapidly into an alpha-helix; but nonlocal HBs usually retard helix formation by "trapping" the polymer in irregular, "balled-up" structures. Most trajectories formed some nonlocal HBs, sometimes as many as eight. But, for (Ala)20, most of these eventually rearranged to form local HBs that lead to alpha-helices. A simple kinetic model describes the rate of converting nonlocal HBs into alpha-helices. Torsional-coordinate MD speeds folding by eliminating bond and angle degrees of freedom and reducing dynamical friction. Thus, the observed 210 ps half-life for helix formation is likely to be a lower bound on the real rate. However, we believe the sequential steps observed here mirror those of real systems.

Thermodynamics of electron transfer in Escherichia coli cytochrome bo3.

The proton translocation mechanism of the Escherichia coli cytochrome bo3 complex is intimately tied to the electron transfers within the enzyme. Herein we evaluate two models of proton translocation in this enzyme, a cytochrome c oxidase-type ion-pump and a Q-cycle mechanism, on the basis of the thermodynamics of electron transfer. We conclude that from a thermodynamic standpoint, a Q-cycle is the more favorable mechanism for proton translocation and is likely occurring in the enzyme.

Evolution of the cytochrome c oxidase proton pump.

The superfamily of quinol and cytochrome c terminal oxidase complexes is related by a homologous subunit containing six positionally conserved histidines that ligate a low-spin heme and a heme-copper dioxygen activating and reduction center. On the basis of the structural similarities of these enzymes, it has been postulated that all members of this superfamily catalyze proton translocation by similar mechanisms and that the CuA center found in most cytochrome c oxidase complexes serves merely as an electron conduit shuttling electrons from ferrocytochrome c into the hydrophobic core of the enzyme. The recent characterization of cytochrome c oxidase complexes and structurally similar cytochrome c:nitric oxide oxidoreductase complexes without CuA centers has strengthened this view. However, recent experimental evidence has shown that there are two ubiquinone(ol) binding sites on the Escherichia coli cytochrome bo3 complex in dynamic equilibrium with the ubiquinone(ol) pool, thereby strengthening the argument for a Q(H2)-loop mechanism of proton translocation [Musser SM et al. (1997) Biochemistry 36:894-902]. In addition, a number of reports suggest that a Q(H2)-loop or another alternate proton translocation mechanism distinct from the mitochondrial aa3-type proton pump functions in Sulfolobus acidocaldarius terminal oxidase complexes. The possibility that a primitive quinol oxidase complex evolved to yield two separate complexes, the cytochrome bc1 and cytochrome c oxidase complexes, is explored here. This idea is the basis for an evolutionary tree constructed using the notion that respiratory complexity and efficiency progressively increased throughout the evolutionary process. The analysis suggests that oxygenic respiration is quite an old process and, in fact, predates nitrogenic respiration as well as reaction-center photosynthesis.

The particulate methane monooxygenase from methylococcus capsulatus (Bath) is a novel copper-containing three-subunit enzyme. Isolation and characterization.

The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivating Methylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl beta-D-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60-80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, alpha, beta, and gamma, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, alpha', beta, and gamma, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The alpha and alpha' subunits from these two forms of the pMMO contain identical N-terminal sequences. The gamma subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12-15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (approximately 10 H+/94-kDa monomeric unit) and eventually become completely inactive.

Reaction of Escherichia coli cytochrome bo3 with substoichiometric ubiquinol-2: a freeze-quench electron paramagnetic resonance investigation.

The reaction of the quinol oxidase cytochrome bo3 from Escherichia coli with ubiquinol-2 (UQ2H2) was carried out using substoichiometric (0.5 equiv) amounts of substrate. Reactions were monitored through the use of freeze-quench EPR spectroscopy. Under 1 atm of argon, semiquinone was formed at the QB site of the enzyme with a formation rate constant of 140 s-1; the QB semiquinone EPR signal decayed with a rate constant of about 5 s-1. Heme b and CuB were reduced within the 10-ms dead time of the freeze-quench experiment and remained at a constant level of reduction over the 1-s time course of the experiment. Quantitation of the reduction levels of QB and heme b during this reaction yielded a reduction potential of 30-60 mV for heme b. Under a dioxygen atmosphere, the rates of semiquinone formation and its subsequent decay were not altered significantly. However, accurate quantitation of the EPR signals for heme b and heme o3 could not be made, due to interference from dioxygen. In the reaction between the QB-depleted enzyme and UQ2H2 under substoichiometric conditions, there was no observable change in the EPR spectra of the enzyme over the time course of the reaction, suggesting an electron transfer from heme b to the binuclear site in the absence of QB which occurs within the dead time of the freeze-quench apparatus. Analysis of the thermodynamics and kinetics of electron transfers in this enzyme suggests that a Q-cycle mechanism for proton translocation is more likely than a cytochrome c oxidase-type ion-pump mechanism.

Kinetic role of electrostatic interactions in the unfolding of hyperthermophilic and mesophilic rubredoxins.

The temperature dependence of the unfolding kinetics of rubredoxins from the hyperthermophile Pyrococcus furiosus (RdPf) and the mesophile Clostridium pasteurianum (RdCp) has been studied. Results show that RdPf unfolds much more slowly, under all experimentally accessible temperature regimes, than RdCp and other typical mesophilic proteins. Rates of RdCp and RdPf unfolding decrease upon increasing the pH above 2 and diverge dramatically at pH 7. As shown by detailed electrostatic energy calculations, this is the result of a differential degree of protonation of the negatively charged amino acids, which causes distinct electrostatic configurations as a function of pH. We propose that ion pairs, particularly those that are placed in key surface positions, may play a kinetic role by mildly clamping the protein and thereby influencing the nature and the number of the vibrational normal modes that are thermally accessible upon unfolding. More generally, these modes are also likely to be affected by the favorable electrostatic configurations, which we have shown to be directly linked to the extremely slow unfolding rates of RdPf at neutral pH. Even at pH 2, in the absence of any salt bridges, the unfolding rates of RdPf are much smaller than those of RdCp. This is ascribed to presently unidentified structural elements of nonelectrostatic nature. Since electrostatic effects influence the unfolding kinetics of both mesophilic and thermophilic rubredoxins, these findings may be of general significance for proteins.

Unfolding mechanism of rubredoxin from Pyrococcus furiosus.

As part of our studies on the structural and dynamic properties of hyperthermostable proteins, we have investigated the unfolding pathways of the small iron-sulfur protein rubredoxin from Pyrococcus furiosus (RdPf) at pH 2. Unfolding has been initiated by temperature jump, triggered by manual mixing of a concentrated protein solution into a thermally preequilibrated buffer. The process has been followed in real time by absorption, tryptophan fluorescence emission, and far-UV circular dichroism. Unlike the case of the mesophilic rubredoxin from Clostridium pasteurianum (RdCp), RdPf displays a complex unfolding kinetics, pointing to the formation of at least three intermediates. All of the steps, including the one involving metal ion release, are extremely slow. However, hydrophobic core relaxation--not Fe3+ loss--is rate-determining for RdPf unfolding. This clearly rules out the fact that Fe3+ is solely responsible for the kinetic stability of RdPf. Results have been discussed in terms of sequential vs parallel pathways, and the possible role of irreversible phenomena has been taken into consideration. Aggregation does not appear to play a significant role in the observed kinetic complexities. According to a proposed sequential mechanism, partial release of secondary structure elements precedes iron loss, which is then followed by further loss of beta-sheet content and, finally, by hydrophobic relaxation. Although the main features of the RdPf unfolding mechanism remain substantially unchanged over the experimentally accessible temperature range, final hydrophobic relaxation gets faster, relative to the other events, as the temperature is decreased. A qualitative assessment of the unfolding activation parameters suggests that this arises from the very low activation energies (Ea) that characterize this step.