Preparation of cytochrome c peroxidase from baker's yeast.

A simple and readily reproducible procedure is presented for the preparation and purification of cytochrome c peroxidase from baker's yeast. Following autolysis of the yeast and extraction, the enzyme is collected on DEAE-cellulose at moderately high ionic strength, cluted, concentrated, and subjected to gel filtration in 0.1 m sodium acetate buffer, pH 5.0. The properties of the crude preparation make gel filtration in this buffer suitable for near-final purification of the heme protein. The enzyme is then easily crystallized by dialysis.     

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.     

Thermal stability of membrane-reconstituted yeast cytochrome c oxidase

The thermal dependence of the structural stability of membrane-reconstituted yeast cytochrome c oxidase has been studied by using different techniques including high-sensitivity differential scanning calorimetry, differential detergent solubility thermal gel analysis, and enzyme activity measurements. For these studies, the enzyme has been reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) vesicles using detergent dialysis. The phospholipid moiety affects the stability of the enzyme as judged by the dependence of the denaturation temperature on the lipid composition of the bilayer. The enzyme is more stable when reconstituted with the 18-carbon, unsaturated phospholipid (DEPC) than with the 14-carbon saturated phospholipid (DMPC). In addition, the shapes of the calorimetric transition profiles are different in the two lipid systems, indicating that not all of the subunits are affected equally by the lipid moiety. The overall enthalpy change for the enzyme denaturation is essentially the same for the two lipid reconstitutions (405 kcal/mol of protein for the DMPC and 425 kcal/mol for the DEPC-reconstituted enzyme). In both systems, the van't Hoff to calorimetric enthalpy ratios are less than 0.2, indicating that the unfolding of the enzyme cannot be represented as a two-state process. Differential detergent solubility experiments have allowed us to determine individual subunit thermal denaturation profiles. These experiments indicate that the major contributors to the main transition peak observed calorimetrically are subunits I and II and that the transition temperature of subunit III is the most affected by the phospholipid moiety. Experiments performed at different scanning rates indicate that the thermal denaturation of the enzyme is a kinetically controlled process characterized by activation energies on the order of 40 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfite binding to a flavodehydrogenase, cytochrome b2 from baker's yeast.

Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone [Capeillère Blandin et al. Eur. J. Biochem. 54, 549--566 (1975)] and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 muM). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavo-proteins may not be as general as previously proposed [Massey et al. J. Biol. Chem. 244, 3999--4006 (1969)].     

Denatured states of yeast cytochrome c induced by heat and guanidinium chloride are structurally and thermodynamically different

A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ? D state, N state ? H state, and H state ? D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.     

Studies on cytochrome c. XI. Circular dichroism studies on synthetic peptides related to the C-terminal region of baker's yeast iso-1-cytochrome c

Circular dichroism studies on synthetic peptides related to the C-terminal region of yeast iso-1-cytochrome c were carried out and compared with conformational studies on horse cytochrome c fragments. Evidence is presented for a weaker predisposition for ordered structure in the former peptides when compared with the corresponding region in horse cytochrome c. These findings agree with theoretical predictions and with observations that yeast and other mammalian type cytochromes c differ in several minor respects.     

The synthesis of cytochrome b on mitochondrial ribosomes in baker's yeast.

Antiserum against a major cytochrome b peptide isolated from yeast mitochondria as described previously (Lin, L.-F.H., and Beattie, D.S., J. Biol. Chem. 1978, 253, 2412--2418) was raised in rabbits and shown to be monospecific against the pure antigen. Mitochondria were isolated from yeast cells grown in [3H]leucine, extracted with Lubrol and treated with antiserum to cytochrome b. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of a single major band of molecular weight 31 000 corresponding to cytochrome b. In order to determine the intracellular site of translation of cytochrome b, yeast cells were labeled in vivo under non-growing conditions with [3H]leucine in the absence or presence of inhibitors of cytoplasmic and mitochondrial protein synthesis. The incorporation of radioactive leucine into the apoprotein of cytochrome b isolated by immunoprecipitation followed by gel electrophoresis was insensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and sensitive to acriflavin, erythromycin, and chloramphenicol (inhibitors of mitochondrial protein synthesis). Furthermore, no cytochrome b apoprotein was present in a cytoplasmic petite mutant which lacked mitochondrial protein synthesis. Cytochrome b is thus a product of protein synthesis on mitochondrial ribosomes.     

Purification and properties of a major cytochrome b peptide from baker's yeast.

A major cytochrome b peptide was purified from yeast mitochondria by a procedure involving solubilization in deoxycholic and cholic acids, ammonium sulfate fractionation, proteolytic digestion, and sucrose gradient centrifugation in the presence of Tween 80. The homogeneity of the purified protein was established by the criteria that the product was spectrally pure and yielded a single band on both sodium dodecyl sulfate polyacrylamide gel electrophoresis, and by gel isoelectric focusing. The purified cytochrome b polypeptide had absorption maxima at 562, 532, and 430 nm in the reduced form and at 525 to 570 nm and 419 nm in the oxidized form. The reduced minus oxidized difference spectra revealed absorption bands at 562, 532, and 430 nm at room temperature and 559, 529, and 429 nm at 77 K, respectively. The heme group was identified as protoheme by formation of the reduced pyridine hemochromogen. Treatment of the reduced form with carbon monoxide affected the absorption spectrum, indicating that the isolated hemoprotein was modified compared to native cytochrome b. The apparent molecular weight of the preparation was 28,000 based on sodium dodecyl sulfate polyacrylamide-gel electrophoresis and 28,800 based on sucrose gradient centrifugation. The isolated cytochrome b polypeptide showed a strong tendency to aggregate.     

Purification and properties of glyoxylate reductase I from baker's yeast.

The purification and properties of NADPH-linked glyoxylate reductase [EC 1. 1. 1. 79] from baker's yeast were studied. Two active fractions (peak I and peak II) were isolated by DEAE-cellulose column chromatography. The peak I fraction was purified to homogeneity by the criteria of disc gel electrophoresis and tentatively designated glyoxylate reductase I. Its molecular weight was calculated to be 31,000 from gel filtration measurements. The enzyme reduced glyoxylate 7 times faster than hydroxypyruvate and was specific for NADPH. The enzyme showed optimum activity between pH 5.5 and 7.2. The Michaelis constants for glyoxylate and NADPH were found to be 13 mM and 4 microM, respectively. The enzymic activity was not significantly affected by anions, except for nitrate and iodide, which were inhibitory.     

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.     

Identification and isolation of the yeast cytochrome c gene.

The iso-1-cytochrome c gene of yeast has been identified and cloned using a synthetic oligodeoxynucleotide as a hybridization probe. The oligomer d[pT-T-A-G-C-A-G-A-A--C-C-G-G] is complementary to a region near the N terminal coding region of the yeast cyc 1 gene. Of several yeast Eco RI fragments which hybridize to this probe, one is changed in size by a G leads to T mutation which eliminates an Eco RI site within the cyc 1 gene. Both the wild-type and the RI- mutant forms were cloned in lambda gt vectors. Maxam-Gilbert sequencing for 91 nucleotides into the coding region for iso-1-cytochrome c yielded a DNA sequence in perfect correspondence with the known protein sequence.     

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.