Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c.

Proton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso-1-cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent-accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in omega-loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent-accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, delta Gd, at 298 K to the free energy change for local unfolding, delta Gop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in delta Gop. Comparisons of delta Gd to delta Gop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high-energy conformations of the reduced form appears to involve the axially coordinated heme.     

Conformational toggling of yeast iso-1-cytochrome C in the oxidized and reduced states

To convert cyt c into a peroxidase-like metalloenzyme, the P71H mutant was designed to introduce a distal histidine. Unexpectedly, its peroxidase activity was found even lower than that of the native, and that the axial ligation of heme iron was changed to His71/His18 in the oxidized state, while to Met80/His18 in the reduced state, characterized by UV-visible, circular dichroism, and resonance Raman spectroscopy. To further probe the functional importance of Pro71 in oxidation state dependent conformational changes occurred in cyt c, the solution structures of P71H mutant in both oxidation states were determined. The structures indicate that the half molecule of cyt c (aa 50-102) presents a kind of "zigzag riveting ruler" structure, residues at certain positions of this region such as Pro71, Lys73 can move a big distance by altering the tertiary structure while maintaining the secondary structures. This finding provides a molecular insight into conformational toggling in different oxidation states of cyt c that is principle significance to its biological functions in electron transfer and apoptosis. Structural analysis also reveals that Pro71 functions as a key hydrophobic patch in the folding of the polypeptide of the region (aa 50-102), to prevent heme pocket from the solvent.     

Assignment of 15N chemical shifts and 15N relaxation measurements for oxidized and reduced iso-1-cytochrome c

A protocol for complete isotopic labeling of iso-1-cytochrome c from the eukaryote Saccharomyces cerevisiae is reported. Assignments are reported for the vast majority of the 15N amide resonances in both oxidized and reduced states. 15N heteronuclear relaxation experiments were collected to study the picosecond-nanosecond backbone dynamics of this protein. Relaxation rates were computed and fit to spectral density functions by a model-free analysis. Backbone amides in the overlapping loop B/C region are the most flexible on the picosecond-nanosecond time scale in both forms of the protein. The results show that, on average, the protein backbone is slightly more dynamic in the oxidized than the reduced state, though not significantly so. Exchange terms, which suggest significant motion on a time scale at least an order of magnitude slower than the overall correlation time of 5.2 ns, were required for only two residues in the reduced state and 27 residues in the oxidized state. When analyzed on a per-residue basis, the lower order parameters found in the oxidized state were scattered throughout the protein, with a few continuous segments found in loop C and the C-terminal helix, suggesting greater flexibility of these regions in the oxidized state. The results provide dynamic interpretations for previously presented structural and functional data, including redox-dependent changes that occur in the protein. The way is now paved for extensive dynamic analysis of variant cytochromes c.     

Characterization of yeast iso-1-cytochrome c mRNA

The iso-1-cytochrome c mRNA has been identified by hybridization of a 32P probe prepared from a plasmid containing the iso-1-cytochrome c gene to RNA size-fractionated on agarose gels and transferred to paper. A hybridization band was visible with RNA prepared from wild type cells, but not with RNA prepared from an iso-1-cytochrome c deletion mutant. RNA prepared from cells containing a nonsense mutation in the iso-1-cytochrome c gene showed reduced levels of hybridization. The RNA that hybridized to the probe was 700 +/- 50 nucleotides in length and was polyadenylated. The cellular levels of this RNA were repressed by glucose, and this repression was achieved within 5 min after glucose addition to a derepressed culture. No precursors of this RNA were detected in wild type cells or in an RNA1 mutant, temperature-sensitive for RNA metabolism. The length of the 3' noncoding region of this RNA was determined to be 200 +/- 25 nucleotides (excluding the poly(A) tail) and the 5' noncoding region was estimated to be about 120 nucleotides in length.     

Sequence of the yeast iso-1-cytochrome c mRNA

The nucleotide sequence of the yeast iso-1-cytochrome c (CYC1) mRNA is presented. The mRNA was enriched by hybridization to cloned CYC1 DNA attached to a solid matrix: either nitrocellulose filters or diazobenzyloxymethyl cellulose powder. The sequence of the 5'-end of the mRNA was determined by the extension of a CYC1-specific dodecanucleotide primer; the sequence of the 3'-end was determined using a decanucleotide d(pT8-G-A) primer. The CYC1 mRNA begins 61 nucleotides 5' to the AUG initiation codon, extends through the coding sequence to 172 to 175 nucleotides 3' to the UAA termination codon, followed by the poly(A) tail. There are no intervening sequences. Some of the sequences that the CYC1 mRNA shares in common with other eukaryotic mRNAs are discussed.     

Interactions between yeast iso-1-cytochrome c and its peroxidase

Isothermal titration calorimetry was used to study the formation of 19 complexes involving yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP). The complexes comprised combinations of the wild-type proteins, six CcP variants, and three Cc variants. Sixteen protein combinations were designed to probe the crystallographically defined interface between Cc and CcP. The data show that the high-affinity sites on Cc and CcP coincide with the crystallographically defined sites. Changing charged residues to alanine increases the enthalpy of complex formation by a constant amount, but the decrease in stability depends on the location of the amino acid substitution. Deleting methyl groups has a small effect on the binding enthalpy and a larger deleterious effect on the binding free energy, consistent with model studies of the hydrophobic effect, and showing that nonpolar interactions also stabilize the complex. Double-mutant cycles were used to determine the coupling energies for nine Cc-CcP residue pairs. Comparing these energies to the crystal structure of the complex leads to the conclusion that many of the substitutions induce a rearrangement of the complex.     

Hydrogen exchange behavior of [U-15N]-labeled oxidized and reduced iso-1-cytochrome c

Heteronuclear NMR spectroscopy was used to measure the hydrogen-deuterium exchange rates of backbone amide hydrogens in both oxidized and reduced [U-15N]iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. The exchange data confirm previously reported data [Marmorino et al. (1993) Protein Sci. 2, 1966-1974], resolve several inconsistencies, and provide more thorough coverage of exchange rates throughout the cytochrome c protein in both oxidation states. Combining the data previously collected on unlabeled C102T with the current data collected on [U-15N]C102T, exchange rates for 53 protons in the oxidized state and 52 protons in the reduced state can now be reported. Most significantly, hydrogen exchange measurements on [U-15N]iso-1-cytochrome c allowed the observation of exchange behavior of the secondary structures, such as large loops, that are not extensively hydrogen-bonded. For the helices, the most slowly exchanging protons are found in the middle of the helix, with more rapidly exchanging protons at the helix ends. The observation for the Omega-loops in cytochrome c is just the opposite. In the loops, the ends contain the most slowly exchanging protons and the loop middles allow more rapid exchange. This is found to be true in cytochrome c loops, even though the loop ends are not attached to any regular secondary structures. Some of the exchange data are strikingly inconsistent with data collected on the C102S variant at a different pH, which suggests pH-dependent dynamic differences in the protein structure. This new hydrogen exchange data for loop residues could have implications for the substructure model of eukaryotic cytochrome c folding. Isotopic labeling of variant forms of cytochrome c can now be used to answer many questions about the structure and folding of this model protein.     

Rational design of a more stable yeast iso-1-cytochrome c.

Yeast iso-1-cytochrome c is one of the least stable mitochondrial cytochromes c. We have used a coordinated approach, combining the known functional and structural properties of cytochromes c, to engineer mutations into yeast iso-1-cytochrome c with the goal of selectively increasing the stability of the protein. The two redox forms of the native protein and six different mutant forms of yeast iso-1-cytochrome c were analyzed by differential scanning calorimetry (DSC). The relative stability, expressed as the difference in the Gibb's free energy of denaturation at a given temperature between the native and mutant forms (DeltaDeltaG(Tref)), was determined for each of the proteins. In both oxidation states, the mutant proteins C102T, T69E/C102T, T96A/C102T, and T69E/T96A/C102T were more stable than the wild-type protein, respectively. The increased stability of the mutant proteins is proposed to be due to the removal of a rare surface cysteine and the stabilization of two distorted alpha-helices.     

Structural gene for yeast iso-2-cytochrome c.

Protein analysis and genetic studies have led to the identification of the structural genes of iso-1-cytochrome c and iso-2-cytochrome c, which constitute, respectively, 95% and 5% of the total amount of cytochrome c in the yeast Saccharomyces cerevisiae. The structural gene CYC1 for iso-1-cytochrome c was previously identified by Sherman et al. (1966) and the structural gene CYC7 for iso-2-cytochrome c is identified in this investigation. A series of the following mutations were selected by appropriate procedures and shown by genetic tests to be allelic: CYC7+ →CYC7-1 →cyc7-1-1 →CYC7-1-1-A, etc., where CYC7 + denotes the wild-type allele determining iso-2-cytochrome c; CYC7-1 denotes a dominant mutant allele causing an approximately 30-fold increase of iso-2-cytochrome c with a normal sequence, and was used as an aid in selecting deficient mutants; cyc7-1-1 denotes a recessive mutant allele causing complete deficiency of iso-2-cytochrome c; and CYC7-1-1-A denotes an intragenic revertant having an altered iso-2-cytochrome c at the same level as iso-2-cytochrome c in the CYC7-1 strains. The suppression of cyc7-1-1 with the known amber suppressor SUP7-a indicated that the defect in cyc7-1-1 was an amber (UAG) nonsense codon. Sequencing revealed a single amino acid replacement of a tyrosine residue for the normal glutamine residue at position 24 in iso-2-cytochrome c from the suppressed cyc7-1-1 strain and also in five revertants of cyc7-1-1, of which three were due to extragenic suppression and two to intragenic reversion. The nature of the mutation that elevated the level of normal iso-2-cytochrome c in the CYC7-1 strain was not identified, although it occurred at or very near the CYC7 locus but outside the translated portion of the gene and it may be associated with a chromosomal aberration. Genetic studies demonstrated that CYC7 is not linked to CYC1, the structural gene for iso-1-cytochrome c.     

Crystallization and preliminary diffraction data for iso-1-cytochrome c from yeast

Deep red crystals of the electron transfer protein, iso-1-cytochrome c from yeast (Saccharomyces cerevisiae), have been obtained from a 90% saturated solution of (NH4)2SO4 containing 2 mg protein/ml, 0.1 M-sodium phosphate and adjusted to pH 6.7. The space group is P4(1)2(1)2 (or P4(3)2(1)2) with a = b = 36.4 A, c = 136.8 A and Z = 8. Crystals are stable for at least ten days in the X-ray beam and diffract to better than 2.0 A resolution. Comparable and morphologically similar crystal forms of three iso-1-cytochrome c mutants at Phe87, a pivotal residue in the electron transport chain, have also been obtained.     

Deletions and replacements of omega loops in yeast iso-1-cytochrome c

omega (omega)-loops are protein secondary structural elements having small distances between segment termini. It should be possible to delete or replace certain of these omega-loops without greatly distorting the overall structure of the remaining portion of the molecule. Functional requirements of regions of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae were investigated by determining the biosynthesis and activity in vivo of mutant forms in which four different omega-loops were individually deleted, or in which one omega-loop was replaced with five different segments. Deletions encompassing amino acid positions 27-33 and 79-83 either prevented synthesis of the holoprotein, or produced highly labile iso-1-cytochromes c, whereas deletions encompassing positions 42-45 and 48-55 allowed partial synthesis and activity. These two latter regions, therefore, are not absolutely required for any biosynthetic process such as heme attachment, mitochondrial import, or for enzymatic interactions. All replacements in Loop A (residue positions 24-33) with same size (10 amino acid residues), longer (13 and 15 amino acid residues), or shorter segments (6 amino acid residues), resulted in strains having at least partial levels of iso-1-cytochrome c; however, the relative activities ranged from zero to almost the normal level. Thus, Loop A does not appear to be essential for such biosynthetic steps as heme attachment and mitochondrial import. In contrast, the full range of relative activities suggest that this region interacts with physiological partners to carry out efficient electron transport.     

Mutagenic specificity: reversion of iso-1-cytochrome c mutants of yeast

In previous studies the nucleotide sequences of numerous mutant codons in the cy1 gene have been identified from altered iso-1-cytochromes c. These studies not only revealed the mutant codons that caused the deficiencies but also experimentally determined which of the base pair changes allowed the formation of functional iso-1-cytochromes c. In this investigation we have quantitatively measured the reversion frequencies of eleven cy1 mutants which were treated with 12 mutagens. The cy1 mutants comprised nine mutants having single-base changes of the AUG initiation codon (Stewart et al., 1971), an ochre mutant cy1–9 (Stewart et al., 1972), and an amber mutant cy1–179 (Stewart &; Sherman, 1972). In some cases the types of induced base changes could be inferred unambiguously from the pattern of reversion. Selective G.C to A.T transitions were induced by ethyl methanesulfonate, diethyl sulfate, N-methyl-N′-nitro-N-nitrosoguanidine, 1-nitrosoimidazolidone-2, nitrous acid, [5-³H]uridine and β-propiolactone. There was no apparent specificity with methyl methanesulfonate, dimethyl sulfate, nitrogen mustard and γ-rays. Ultraviolet light induced high rates of reversion of the ochre and amber mutants, but in these instances it appears as if the selective action is due to particular nucleotide sequences and not due to simple types of base pair changes.     

High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c

The structure of yeast iso-1-cytochrome c has been refined against X-ray diffraction data to a nominal resolution of 1.23 A. The atomic model contains 893 protein atoms, as well as 116 water molecules and one sulfate anion. Also included in the refinement are 886 hydrogen atoms belonging to the protein molecule. The crystallographic R-factor is 0.192 for the 12,513 reflections with F greater than or equal to 3 sigma (F) in the resolution range 6.0 to 1.23 A. Co-ordinate accuracy is estimated to be better than 0.18 A. The iso-1-cytochrome c molecule has the typical cytochrome c fold, with the polypeptide chain organized into a series of alpha-helices and reverse turns that serve to envelop the heme prosthetic group in a hydrophobic pocket. Inspection of the conformations of helices in the molecule shows that the local environments of the helices, in particular the presence of intrahelical threonine residues, cause distortions from ideal alpha-helical geometry. Analysis of the internal mobility of iso-1-cytochrome c, based on refined crystallographic temperature factors, shows that the most rigid parts of the molecule are those that are closely associated with the heme group. The degree of saturation of hydrogen-bonding potential is high, with 90% of all polar atoms found to participate in hydrogen bonding. The geometry of intramolecular hydrogen bonds is typical of that observed in other high-resolution protein structures. The 116 water molecules present in the model represent about 41% of those expected to be present in the asymmetric unit. The majority of the water molecules are organized into a small number of hydrogen-bonding networks that are anchored to the protein surface. Comparison of the structure of yeast iso-1-cytochrome c with those of tuna and rice cytochromes c shows that these three molecules have very high structural similarity, with the atomic packing in the heme crevice region being particularly highly conserved. Large conformational differences that are observed between these cytochromes c can be explained by amino acid substitutions. Additional subtle differences in the positioning of the side-chains of several highly conserved residues are also observed and occur due to unique features in the local environments of each cytochrome c molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystallization of yeast iso-2-cytochrome c using a novel hair seeding technique

A hair seeding technique has been developed to obtain diffraction quality crystals of yeast (Saccharomyces cerevisiae) iso-2-cytochrome c, a model for studies of protein folding and biological electron transfer reactions. Deep red crystals of this protein were obtained from 88 to 92% saturated solutions of ammonium sulfate containing 20 mg protein/ml, 0.1 M-sodium phoshate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to phosphate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to pH 6.0. Rapid crystal growth was observed, but only along the path of the seeding hair stroke. The space group is P4(3)2(1)2 (or P4(1)2(1)2) with a = b = 36.4 A, c = 137.8 A (1 A = 0.1 nm) and Z = 8. Crystals are stable in the X-ray beam for more than 10 days and diffract to at least 2.5 A resolution. The same hair seeding methodology has proven useful in obtaining crystals of specifically designed mutant iso-2 proteins and in other protein systems where consistent crystal growth had previously proven difficult to attain.     

Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.     

Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c

The structural and folding requirements of eukaryotic cytochromes c have been investigated by determining the appropriate DNA sequences of a collection of 46 independent cyc 1 missense mutations obtained in the yeast Saccharomyces cerevisiae and by deducing the corresponding amino acid replacements that abolish function of iso-1-cytochrome c. A total of 33 different replacements at 19 amino acid positions were uncovered in this and previous studies. Because all of these nonfunctional iso-1-cytochromes c are produced at far below the normal level and because a representative number are labile in vitro, most of the replacements appear to be affecting stability of the protein or heme attachment. By considering the tertiary structure of related cytochromes c, the loss of function of most of the mutant iso-1-cytochromes c could be attributed to either replacements of critical residues that directly interact with the heme group or to replacements that disrupt the proper folding of the protein. The replacements of residues interacting with the heme group include those required for covalent attachment (Cys-19 and Cys-22), ligand formation (His-23 and Met-85), and formation of the immediate heme environment (Leu-37, Tyr-53, Trp-64, and Leu-73). Proper folding of the protein is prevented by replacements of glycine residues at sites that cannot accommodate side chains (Gly-11 and Gly-34); by replacements of residues with proline, which limit the torsion angle (Leu-14 and His-38); and by replacements apparently unable to direct the local folding of the backbone into the proper conformation (Pro-35, Tyr-72, Asn-75, Pro-76, Lys-84, Leu-99, and Leu-103). Even though most of the missense mutations occurred at sites corresponding to evolutionarily invariant or conserved residues, a consideration of the replacements in functional revertants indicates that the requirement for residues evolutionarily preserved is less stringent than commonly assumed.     

An extensive deletion causing overproduction of yeast iso-2-cytochrome c

CYC7-H3 is a cis-dominant regulatory mutation that causes a 20-fold overproduction of yeast iso-2-cytochrome c. The CYC7-H3 mutation is an approximately 5 kb deletion with one breakpoint located in the 5' noncoding region of the CYC7 gene, approximately 200 base from the ATG initiation codon. The deletion apparently fuses a new regulatory region to the structural portion of the CYC7 locus. The CYC7-H3 deletion encompasses the RAD23 locus, which controls UV sensitivity and the ANP1 locus, which controls osmotic sensitivity. The gene cluster CYC7-RAD23-ANP1 displays striking similarity to the gene cluster CYC1-OSM1-RAD7, which controls, respectively, iso-1-cytochrome c, osmotic sensitivity and UV sensitivity. We suggest that these gene clusters are related by an ancient transpositional event.     

Effect of enzymatic methylation of yeast iso-1-cytochrome c on its isoelectric point

Yeast iso-1- unmethylated and methylated apocytochrome c were synthesized in vitro by translating yeast cytochrome c mRNA, and by subsequently methylating the protein product. Unmethylated and methylated iso-1-holocytochrome c were extracted from Saccharomyces cerevisiae. By employing a column isoelectrofocusing technique, the pI values of these proteins were determined. The pI values of unmethylated and methylated apocytochrome c were found to be 9.60 and 8.70, respectively, with a difference of 0.90 pI unit. On the other hand, the pI values of unmethylated and methylated holocytochrome c were 9.72 and 9.68, respectively, with a difference of 0.04 unit. Therefore, although the pI values of both apo- and holocytochrome c decreased by methylation, methylation of apocytochrome c had a more profound effect on the pI of the protein. The result also indicated that conjugation of heme to apocytochrome c increased its pI value, resulting in the more "compact" and basic structure of the protein. The observed magnitude of the pI change subsequent to the methylation of apocytochrome c (decrease of 0.90 unit) seemed to be contradictory to the predicted increase in the value, since the positive charge is fixed on the quaternary amino group of trimethyllysine and there is no proton to titrate. Trimethylation of epsilon-NH2 group of Res-72 lysine of apocytochrome c could disrupt any possible hydrogen bond formed by the nitrogen atom of Res-72 lysine residues, as visualized by a space-filling model. The model and observed shift in the "effective charge" of the protein strongly suggest that conformational change in the apoprotein takes place upon methylation. This presumably altered conformation along with the decrease in pI caused by methylation may play a role in enhancement of apocytochrome c import into mitochondria.