Crystal structure of Leishmania major peroxidase and characterization of the compound i tryptophan radical

The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX). One common feature is a key Trp residue, Trp(208) in LmP and Trp(191) in CCP, that is situated adjacent to the proximal His heme ligand in CCP, APX, and LmP. In CCP, Trp(191) forms a stable cationic radical after reaction with H(2)O(2) to form Compound I; in APX, the radical is located on the porphyrin ring. In order to clarify the role of Trp(208) in LmP and to further probe peroxidase structure-function relationships, we have determined the crystal structure of LmP and have studied the role of Trp(208) using electron paramagnetic resonance spectroscopy (EPR), mutagenesis, and enzyme kinetics. Both CCP and LmP have an extended section of β structure near Trp(191) and Trp(208), respectively, which is absent in APX. This region provides stability to the Trp(191) radical in CCP. EPR of LmP Compound I exhibits an intense and stable signal similar to CCP Compound I. In the LmP W208F mutant, this signal disappears, indicating that Trp(208) forms a stable cationic radical. In LmP conversion of the Cys(197) to Thr significantly weakens the Compound I EPR signal and dramatically lowers enzyme activity. These results further support the view that modulation of the local electrostatic environment controls the stability of the Trp radical in peroxidases. Our results also suggest that the biological role of LmP is to function as a cytochrome c peroxidase.     

Role of proximal methionine residues in Leishmania major peroxidase

The active site architecture of Leishmania major peroxidase (LmP) is very similar with both cytochrome c peroxidase and ascorbate peroxidase. We utilized point mutagenesis to investigate if the conserved proximal methionine residues (Met248 and Met249) in LmP help in controlling catalysis. Steady-state kinetics of methionine mutants shows that ferrocytochrome c oxidation is <2% of wild type levels without affecting the second order rate constant of first phase of Compound I formation, while the activity toward a small molecule substrate, guaiacol or iodide, increases. Our diode array stopped-flow spectral studies show that the porphyrin π-cation radical of Compound I in mutant LmP is more stable than wild type enzyme. These results suggest that the electronegative sulfur atoms of the proximal pocket are critical factors for controlling the location of a stable Compound I radical in heme peroxidases and are important in the oxidation of ferrocytochrome c.     

Cation-induced stabilization of the engineered cation-binding loop in cytochrome c peroxidase (CcP)

We have previously shown that the K(+) site found in the proximal heme pocket of ascorbate peroxidase (APX) could be successfully engineered into the closely homologous cytochrome c peroxidase (CcP) [Bonagura et al., (1996) Biochemistry 35, 6107-6115; Bonagura et al. (1999) Biochemistry 38, 5538-5545]. In addition, specificity could be switched to binding Ca(2+) as found in other peroxidases [Bonagura et al. (1999) J. Biol. Chem. 274, 37827-37833]. The introduction of a proximal cation-binding site also promotes conversion of the Trp191 containing cation-binding loop from a "closed" to an "open" conformer. In the present study we have changed a crucial hinge residue of the cation-binding loop, Asn195, to Pro which stabilizes the loop, albeit, only in the presence of bound K(+). The crystal structure of this mutant, N195PK2, has been refined to 1.9 A. As predicted, introduction of this crucial hinge residue stabilizes the cation-binding loop in the presence of the bound K(+). As in earlier work, the characteristic EPR signal of Trp191 cation radical becomes progressively weaker with increasing [K(+)] and the lifetime of the Trp191 radical also has been considerably shortened in this mutant. This mutant CcP exhibits reduced enzyme activity, which could be titrated to lower levels with increasing [K(+)] when horse heart cytochrome c is the substrate. However, with yeast cytochrome c as the substrate, the mutant was as active as wild-type at low ionic strength, but 40-fold lower at high ionic strength. We attribute this difference to a change in the rate-limiting step as a function of ionic strength when yeast cytochrome c is the substrate.     

Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase.

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.     

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.     

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.     

The structure of an electron transfer complex containing a cytochrome c and a peroxidase

Efficient biological electron transfer may require a fluid association of redox partners. Two noncrystallographic methods (a new molecular docking program and 1H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) of Paracoccus denitrificans and cytochromes c. For the natural redox partner, cytochrome c550, the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase. In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase. These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility.     

Cytochrome c reactivity in its complexes with mammalian cytochrome c oxidase and yeast peroxidase

1.

1. The ascorbate reducibility of cytochrome c (beef or horse heart) in its complexes with cytochrome c oxidase (beef heart) and cytochrome c peroxidase (yeast) has been studied.     

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.     

Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.     

Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants

Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme that is specific to plants and algae and is indispensable to protect chloroplasts and other cell constituents from damage by hydrogen peroxide and hydroxyl radicals produced from it. In this review, first, the participation of ascorbate peroxidase in the scavenging of hydrogen peroxide in chloroplasts is briefly described. Subsequently, the phylogenic distribution of ascorbate peroxidase in relation to other hydrogen peroxide-scavenging peroxidases using glutathione, NADH and cytochrome c is summarized. Chloroplastic and cytosolic isozymes of ascorbate peroxidase have been found, and show some differences in enzymatic properties. The basic properties of ascorbate peroxidases, however, are very different from those of the guaiacol peroxidases so far isolated from plant tissues. Amino acid sequence and other molecular properties indicate that ascorbate peroxidase resembles cytochrome c peroxidase from fungi rather than guaiacol peroxidase from plants, and it is proposed that the plant and yeast hydrogen peroxide-scavenging peroxidases have the same ancestor.     

Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c.

Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.     

Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T.L., & Finzel, B.C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the three cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign. For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amino acid sequences of the two heme c-binding sites of Pseudomonas cytochrome-c peroxidase

The amino acid sequences of the two heme c-containing tryptic peptides of Pseudomonas cytochrome-c peroxidase have been determined. The tryptic peptides were isolated from two cyanogen bromide fragments of the protein. Both heme-binding sites have the Cys-X-Y-Cys-His structure characteristic of c-type cytochromes. The sequences of the two peptides show distinct homology with each other, suggesting the occurrence of gene doubling during evolution of the protein molecule. The function of the heme c moieties in the catalytic cycle of the enzyme is discussed on the basis of their homology with the proximal histidine region of peroxidase (horseradish peroxidase and yeast cytochrome-c peroxidase) and cytochromes (horse cytochrome c and Pseudomonas cytochrome c-551).     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.     

Cocrystals of yeast cytochrome c peroxidase and horse heart cytochrome c

Yeast cytochrome c peroxidase and horse heart cytochrome c have been cocrystallized in a form suitable for x-ray diffraction studies and the structure determined at 3.3 A. The asymmetric unit contains a dimer of the peroxidase which was oriented and positioned in the unit cell using molecular replacement techniques. Similar attempts to locate the cytochrome c molecules were unsuccessful. The peroxidase dimer model was subjected to eight rounds of restrained parameters least squares refinement after which the crystallographic R factor was 0.27 at 3.3 A. Examination of a 2Fo-Fc electron density map showed large "empty" regions between peroxidase dimers with no indication of cytochrome c molecules. Electrophoretic analysis of the crystals demonstrated the presence of the peroxidase and cytochrome c in an approximate equal molar ratio. Therefore, while cytochrome c molecules are present in the unit cell they are orientationally disordered and occupy the space between peroxidase dimers.     

Electrostatic control of the tryptophan radical in cytochrome c peroxidase

Previously a K(+)-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K(+) influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Trp191 cation radical is dependent on the occupancy of the engineered K(+) site and that the Trp191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Trp191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 A from the Trp radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Trp radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Trp radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Trp191 radical.     

On the specificity of cytochrome c synthetase in recognition of the amino acid sequence of apocytochrome c

Two forms of yeast cytochrome c synthetases with different specificities were resolved, one (synthetase I), solubilized from mitochondria or the cell debris with Triton X-100, recognizing not horse apocytochrome c but yeast apo-iso-1-cytochrome c as a substrate and the other (synthetase II) still bound with the particulate fraction from mitochondria after treatment with Triton, recognizing both horse and yeast apocytochromes c. The activity with labeled yeast apo-iso-1-cytochrome c as a substrate of cytochrome c synthetase I can be quantitatively inhibited by nonlabeled Candida krusei apocytochrome c and partially by nonlabeled tuna apocytochrome c but not by nonlabeled horse apocytochrome c indicating a specific amino acid sequence being recognized. However, an enzyme similarly solubilized from beef heart mitochondria recognized both horse apocytochrome c and yeast apo-iso-1-cytochrome c for attachment of heme. In view of the fact that the yeast synthetase II and the beef synthetase can both utilize either horse apocytochrome c or yeast apo-iso-1-cytochrome c as substrates, we suggest that these enzymes may also be involved in biosynthesis of cytochrome c1, that is, the ability to attach heme to apocytochrome c and apocytochrome c1 may have been conserved in eucaryotic cells, and that both synthetases may therefore be homologous.     

Species-specific differences in covalently crosslinked complexes of yeast cytochrome C peroxidase with horse and yeast ISO-1 ferricytochromes C

• 1.1. The results of chemically crosslinking yeast cytochrome c peroxidase with both horse and yeast iso-1 ferricytochromes c have been studied by a combination of gel electrophoresis and proton NMR spectroscopy.
• 2.2. The complexes were formed at a variety of potassium phosphate concentrations ranging from 10 to 300 mM using the water soluble crosslinking agent, EDC (l-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide).
• 3.3. The primary crosslinking product in both cases is the 1:1 covalent complex, but, for each pair of partner proteins the yield of the 1:1 crosslinked complex varies with the salt concentration.
• 4.4. Furthermore, at low salt concentrations the yield of the 1:1 covalent complex involving horse cytochrome c is much larger than the yield of the 1:1 covalent complex formed with yeast iso-1 cytochrome c, whereas at high salt concentrations the situation is reversed.
• 5.5. Proton NMR spectroscopy, in combination with gel electrophoresis, provides evidence for the formation of different types of 1:1 complexes for the peroxidase/yeast cytochrome c pair and has been used to study the effect of changes in the solution ionic strength upon both the peroxidases/horse cytochrome c and the peroxidase/yeast cytochrome c complexes.
• 6.6. This work indicates that electrostatic interactions between proteins play a dominant role in formation of complexes between cytochrome c peroxidase and horse ferricytochrome c, whereas the hydrophobic effect plays a comparatively larger role in stabilizing complexes between cytochrome c peroxidase and yeast iso-1 ferricytochrome c.