Conformational relaxation in hemoproteins: the cytochrome P-450cam case

Photodissociation of (CO)P-450(cam)(substrate) complexes was found to trigger a conformational relaxation process that interferes with ligand rebinding at temperatures as low as 140 K even though the protein conformational substates (CS(1)) remain frozen. To analyze the rebinding and relaxation kinetics, we developed a model that takes the distribution of relaxation rates explicitly into account and in which rebinding and relaxation rates are connected by a linear free energy relation. In all complexes heme relaxation occurs first and is probably faster than 100 ns even at 77 K. This is the only process found in substrate-free P-450(cam). Above 140 K and in the presence of a substrate, this initial, fast rebinding state (P) progressively relaxes to another state (P degrees ) in which rebinding is slower. The relaxation rate is independent of solvent rigidity and is governed by the protein's internal dynamics. Rebinding enthalpies in P and P degrees as well as the enthalpy shift brought about by relaxation correlate with the substrate propensity to block access to the iron site. In P degrees the barrier is higher because the substrate is closer to the heme normal and exerts more steric repulsion for CO binding. The relaxation process implies the return of substrate and heme to their ligand-free positions in which access to the heme is reduced.     

Structural transitions of carboxymethylated cytochrome c: calorimetric and circular dichroic studies

Circular dichroism and differential scanning calorimetry studies on the unfolding-refolding process of native and carboxymethylated cytochrome c, induced either by temperature or chemical agents, have been performed. The results have shown that the modified protein has a decreased conformational stability with respect to the native state, in agreement with a structure less compact, but still highly folded, which behaves as a thermodynamically stable "intermediate" between native and fully unfolded cytochrome c.     

Investigating the local flexibility of functional residues in hemoproteins

It is now widely accepted that protein function depends not only on structure, but also on flexibility. However, the way mechanical properties contribute to catalytic mechanisms remains unclear. Here, we propose a method for investigating local flexibility within protein structures that combines a reduced protein representation with Brownian dynamics simulations. An analysis of residue fluctuations during the dynamics simulation yields a rigidity profile for the protein made up of force constants describing the ease of displacing each residue with respect to the rest of the structure. This approach has been applied to the analysis of a set of hemoproteins, one of the functionally most diverse protein families. Six proteins containing one or two heme groups have been studied, paying particular attention to the mechanical properties of the active-site residues. The calculated rigidity profiles show that active site residues are generally associated with high force constants and thus rigidly held in place. This observation also holds for diheme proteins if their mechanical properties are analyzed domain by domain. We note, however, that residues other than those in the active site can also have high force constants, as in the case of residues belonging to the folding nucleus of c-type hemoproteins.     

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.     

Reaction of hydrogen peroxide and peroxidase activity in carboxymethylated cytochrome c: spectroscopic and kinetic studies

The peroxidase activity of carboxymethylated cytochrome c (Cmcytc) has been investigated by spectroscopic and kinetic techniques to examine the effect of carboxymethylation on the peroxidase activity of native cytochrome c (cytc). The optical spectrum suggests that the reaction of Cmcytc with H(2)O(2) proceeds through only one intermediate, compound I. The apparent rate constant (k(app)) for the reaction was found to be 17, 72 and 210 M(-1) s(-1) at pH 7.0, 5.0 and 3.5 respectively. These values are about 60 times larger than those reported for native cytc (0.236 M(-1) s(-1) at pH 7.0), and about five orders of magnitude lower than those for classical peroxidases. Cmcytc was found to catalyse oxidation of organic and inorganic substrates. The second order rate constant for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) by Cmcytc (205 [H(2)O(2)] s(-1)) is found to be larger than the corresponding value for native cytc (50 [H(2)O(2)] s(-1)) at pH 6.0. The carboxymethylation of cytc ruptures the Fe-S (Met 80) bond and increases the rate of its reaction with H(2)O(2), and its catalytic activity. The specific activity of Cmcytc was measured spectrophotometrically by the reported method using ABTS as substrate, and was found to be 288, 473 and 872 microM min(-1) mg(-1) at pH 7.0, 5.0 and 3.5 respectively. Resonance Raman studies indicated the presence of a bis-histidine coordinated form of Cmcytc at neutral pH, and the existence of a population distribution of different ligation states such as bis-histidine (HH), histidine-water (HW) and five coordinate (5C) forms at lower pH. The relative population of different species in Cmcytc was found to be HH (approximately 100%, approximately 50%, approximately 44%), HW (approximately 0%, approximately 44%, 41%) and 5C (approximately 0%, approximately 6%, 15%) at pH 7.0, 4.7 and 3.1 respectively. We have attempted to correlate the pH dependence of the reaction of Cmcytc with hydrogen peroxide and its peroxidase activity with the haem stereochemical structures observed for Cmcytc. Steady-state and time-resolved tryptophan fluorescence studies on Cmcytc were done to probe the conformational changes around the haem pocket of Cmcytc.     

Peroxynitrite detoxification by horse heart carboxymethylated cytochrome c is allosterically modulated by cardiolipin

Carboxymethylation of equine heart cytochrome c (cytc) changes its tertiary structure by disrupting the heme-Fe-Met80 distal bond, such that carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) on peroxynitrite isomerization by ferric CM-cytc (CM-cytc-Fe(III)) is reported. Unlike native ferric cytc (cytc-Fe(III)), CM-cytc-Fe(III) catalyzes peroxynitrite isomerization, the value of the second order rate constant (k_on) is 6.8 × 10⁴ M⁻¹ s⁻¹. However, CM-cytc-Fe(III) is less effective in peroxynitrite isomerization than CL-bound cytc-Fe(III) (CL-cytc-Fe(III); k_on = 3.2 × 10⁵ M⁻¹ s⁻¹). Moreover, CL binding to CM-cytc-Fe(III) facilitates peroxynitrite isomerization (k_on = 5.3 × 10⁵ M⁻¹ s⁻¹). Furthermore, the value of the dissociation equilibrium constant for CL binding to CM-cytc-Fe(III) (K = 1.8 × 10⁻⁵ M) is lower than that reported for CL-cytc-Fe(III) complex formation (K = 5.1 × 10⁻⁵ M). Although CM-cytc-Fe(III) and CL-cytc-Fe(III) display a different heme distal geometry and heme-Fe(III) reactivity, the heme pocket and the CL cleft are allosterically linked.     

Urea-induced modification of cytochrome c flexibility as probed by cyanide binding

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant k_a increased 20-fold when the concentration of urea was raised from 0 M to 6 M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the pK of acid-induced unfolding, the k_a value reached its maximum (k_a,max) in all urea concentrations. Interestingly, the k_a,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe–S(Met80) bond disruption and for ligand binding to heme iron.     

Structure-function relationship in hemoproteins: The role of cytochrome c 3 in the reduction of colloidal sulfur by sulfate-reducing bacteria

Cytochromes c ₃ of different strains of sulfatereducing bacteria have been purified and tested for their capacity to reduce colloidal sulfur to hydrogen sulfide. The results are in good agreement with the activities reported for the whole cells. Cytochrome c ₃ is the sulfur reductase of some strains of sulfate-reducing bacteria such as Desulfovibrio desulfuricans Norway 4 and sulfate-reducing bacterium strain 9974 from which the sulfur reductase activity can be purified with the cytochrome c ₃. In contrast, Desulfovibrio vulgaris Hildenborough cytochrome c ₃ is inhibited by the product of the reaction namely hydrogen sulfide. Chloramphenicol has no effect on the sulfur reductase activity of D. desulfuricans Norway 4 when resting cells grown on lactate-sulfate medium are put in the presence of colloidal sulfur. This shows that the sulfur reductase activity is constitutive and corresponds to the fact that colloidal sulfur grown cells do not contain more cytochrome c ₃ (or another sulfur reductase) than lactate-sulfate-grown cells.     

Effect of steady illumination on the binding of carbon monoxide by carboxymethylated cytochrome c (Short Communication)

The quantum yield, [unk], is reported for the photodissociation of CO from reduced carboxymethylated cytochrome c. The values of [unk] obtained are low relative to that for myoglobin and are pH-independent, being 0.23 at pH6.1 and 0.27 at pH9.7.     

Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b,iron-sulfur protein and cytochrome c1

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc₁. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Q_o site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c₁, iii) reduction of cytochrome c₁ by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c₁.In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c₁ and cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme c₁ of cytochrome c₁, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of cytochrome bc₁ were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Q_o site and cytochrome c₁ helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Q_o site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.     

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide.

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant. (Formula: see text.) Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552, + 0.19 V, is markedly less than that of horse heart cytochrome c. (2) deltaHox of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) deltaSox and deltaSred of cytochrome c-552 are more negative than those of horse heart cytochrome c. (4) kred of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.     

Inhibition of the cytochrome c/cytochrome c oxidase system by cytochrome c derivatives and related fragments

The oxidation of ferrocytochrome c mediated by cytochrome c oxidase was investigated in the presence of ferricytochrome c, trifluoroacetyl-cytochrome c, the heme fragments Hse65-[1-65] and Hse80-[1-80] and their respective porphyrin derivatives, as well as carboxymethylated apoprotein and related fragments, polycations, salts and neutral additives. The inhibition of the redox reaction by salts and neutral molecules, even if in theoretical agreement with their effect on electrostatic interactions, may alternatively be interpreted in terms of hydrophobicity. The latter can account for the inhibitory properties of trifluoroacetylated ferricytochrome c, similar to those of ferricytochrome c. On the assumption that the inhibitory properties of some of the investigated derivatives monitor their binding affinities to the cytochrome c oxidase at the cytochrome c binding sites, the experimental results do not confirm a primarily electrostatic character for the cytochrome c/cytochrome c oxidase association process. Strong indication was found that the cytochrome c C-terminal sequence is critically involved in the complex formation. Conformational studies by circular dichroism measurements and IR spectroscopy in solution and in solid state respectively, show that some of the derivatives examined may possibly acqkuire in the binding process to the oxidase, as secondary structure similar to that present in the native cytochrome c.     

Kinetic studies on redox reactions of hemoproteins. II. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ascorbic acid.

The reductions of thermoresistant cytochrome c-552 and horse heart cytochrome c by ascorbic acid were studied by the stopped-flow method between pH 4 and 10. The results were as follows (1) The reduction of horse heart cytochrome c showed two relaxation decays above pH 8.5, one of which was pseudo-first order, as was the case below pH 8, while the other was nearly concentration-independent. These results were consistent with those reported by Greenwood and Palmer (J. Biol. Chem. (1965) 240, 3660-3663). (2) For the reduction of cytochrome c-552, only a single relaxational decay that obeyed pseudo-first order kinetics was observed. (3) It seems most reasonable to assume that the concentration-independent relaxation process can be attributed to the isomerization reaction accompanying ligand exchange, since it is known that only horse heart cytochrome c exhibits ligand exchange, involving a residue with pK 9.3.     

Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase

The reactions of horse heart cytochrome c with succinate-cytochrome c reductase and cytochrome oxidase were studied as a function of ionic strength using both spectrophotometric and oxygen electrode assay techniques. The kinetic parameter Vmax/Km for both reactions decreased very rapidly as the ionic strength was increased, indicating that electrostatic interactions were important to the reactions. A new semiempirical relationship for the electrostatic energy of interaction between cytochrome c and its oxidation-reduction partners was developed, in which specific complementary charge-pair interactions between lysine amino groups on cytochrome c and negatively charged carboxylate groups on the other protein are assumed to dominate the interaction. The contribution of individual cytochrome c lysine amino groups to the electrostatic interaction was estimated from the decrease in reaction rate caused by specific modification of the lysine amino groups by reagents that change the charge to 0 or -1. These estimates range from -0.9 kcal/mol for lysines immediately surrounding the heme crevice of cytochrome c to 0 kcal/mol for lysines well removed from the heme crevice region. The semiempirical relationship for the total electrostatic energy of interaction was in quantitative agreement with the experimental ionic strength dependence of the reaction rates when the parameters were based on the specific lysine modification results. The electrostatic energies of interaction between cytochrome c and its reductase and oxidase were nearly the same, providing additional evidence that the two reactions take place at similar sites on cytochrome c.