pH-induced conformation changes and equilibrium unfolding in yeast iso-2 cytochrome c

The relationship between pH-induced conformational changes in iso-2 cytochrome c from Saccharomyces cerevisiae and the guanidine hydrochloride induced unfolding transition has been investigated. Comparison of equilibrium unfolding transitions at acid, neutral, and alkaline pH shows that stability toward guanidine hydrochloride denaturation is decreased at low pH but increased at high pH. In the acid range the decrease in stability of the folded protein is correlated with changes in the visible spectrum, which indicate conversion to a high-spin heme state--probably involving the loss of heme ligands. The increase in stability at high pH is correlated with a pH-induced conformational change with an apparent pK near 8. As in the case of homologous cytochromes c, this transition involves the loss of the 695-nm absorbance band with only minor changes in other optical parameters. For the unfolded protein, optical spectroscopy and 1H NMR spectroscopy are consistent with a random coil unfolded state in which amino acid side chains serve as (low-spin) heme ligands at both neutral and alkaline pH. However, the paramagnetic region of the proton NMR spectrum of unfolded iso-2 cytochrome c indicates a change in the (low-spin) heme-ligand complex at high pH. Apparently, the folded and unfolded states of the (inactive) alkaline form differ from the corresponding states of the less stable native protein.     

Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c

The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c.     

The structure and function of omega loop A replacements in cytochrome c.

The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C. This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.     

Yeast iso-1-cytochrome c. A 2.8 A resolution three-dimensional structure determination

A molecular replacement approach, augmented with the results of predictive modeling procedures, solvent accessibility studies, packing analyses and translational coefficient searches, has been used to elucidate the 2.8 A (1 A = 0.1 nm) resolution structure of yeast iso-1-cytochrome c. An examination of the polypeptide chain folding of this protein shows it to have unique conformations in three regions, upon comparison with the structures of other eukaryotic cytochromes c. These include: residues -5 to +1 at the N-terminal end of the polypeptide chain, which are in an extended conformation and project in large part off the surface of the protein; residues 19 to 26, which form a surface beta-loop on the His18 ligand side of the central heme group; and, the C-terminal end of the helical segment composed of residues 49 to 56, which serves to form a part of the heme pocket. Structural studies also show that the highly reactive sulfhydryl group of Cys102 is buried within a hydrophobic region in the monomer form of yeast iso-1-cytochrome c. Dimerization of yeast iso-1-cytochrome c through disulfide bond formation between two such residues would require a substantial conformational change in the C-terminal helix of this protein. Another unique structural feature, the trimethylated side-chain of Lys72, is located on the surface of yeast iso-1-cytochrome c near the solvent-exposed edge of the bound heme prosthetic group. On the basis of the results of these and other structural studies, an analysis of the spatial conservation of structural features in the heme pocket of eukaryotic cytochromes c has been conducted. It was found that the residues involved could be divided into three general classes. The current structural analyses and additional modeling studies have also been used to explain the altered functional properties observed for mutant yeast iso-1-cytochrome c proteins.     

Construction and characterization of mutant iso-2-cytochromes c with replacement of conserved prolines

Oligonucleotide-directed mutagenesis has been used to construct two mutant forms of iso-2-cytochrome c. In one, Pro-30 is replaced by threonine; in the other, Pro-76 is replaced by glycine. Both prolines are fully conserved among mitochondrial cytochromes c and play important structural and functional roles. Yeast with either the Pro-30 or the Gly-76 mutation has appreciable levels of mutant protein in vivo and grows on media containing nonfermentable carbon sources. Thus, neither mutation blocks protein targeting to mitochondria, uptake by mitochondria, covalent attachment of heme, or in vivo function. As judged by ultraviolet-visible spectrophotometry and proton nuclear magnetic resonance spectroscopy, the nativelike conformation of purified Gly-76 iso-2 at pH 6 is almost indistinguishable from that of the normal protein at pH 6. Ultraviolet second-derivative spectrophotometry, however, suggests an increase in the average number of exposed tyrosine side chains, with 2.25 out of 5 residues exposed for the mutant compared to 1.95 for normal iso-2. Above neutral pH, the protein folds to a mutant conformation possibly related to alkaline cytochrome c. Nuclear Overhauser difference spectroscopy of the reduced nativelike conformation allows assignment of several proton resonances and comparison of side-chain conformations of the heme ligand Met-80 in the mutant and the normal proteins. The proton chemical shifts for the assigned resonances are the same within errors for Gly-76 iso-2 and normal iso-2 at pD 6, 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple low spin forms of the cytochrome c ferrihemochrome. EPR spectra of various eukaryotic and prokaryotic cytochromes c.

1. Despite the same methionine-sulfur:heme-iron:imidazole-nitrogen hemochrome structure observed by x-ray crystallography in four of the seven c-type eukaryotic and prokaryotic cytochromes examined, and the occurrence of the characteristic 695 nm absorption band correlated with the presence of a methionine-sulfur:heme-iron axial ligand in all seven proteins, they fall into two distinct classes on the basis of their EPR and optical spectra. The horse, tuna, and bakers' yeast iso-1 cytochromes c have a predominant neutral pH EPR form with g1=3.06, g2=2.26, and g3=1.25, while the bakers' yeast iso-2 and Euglena cytochromes c, the Rhodospirillum rubrum cytochrome c2, and the Paracoccus denitrificans cytochrome c550 all have a predominant neutral pH EPR form with g1=3.2, g2=2.05, and g3=1.39. The ferricytochromes with g1=3.06 have a B-Q splitting that is approximately 150 cm-1 larger than the ferricytochromes with g1=3.2. 2. Each of the cytochromes displays up to four low spin EPR forms that are in pH-dependent equilibrium and can all be observed at near neutral pH. As the pH is raised the predominant neutral pH form is converted into two forms with g1=3.4 and g1=3.6, identified by comparsion with model compounds and other heme proteins as epsilon-amino:heme-iron:imidazole and bis-epsilon-amino:heme-iron ferrihemochromes, respectively. 3. The pK for the conversion of the predominant neutral pH EPR form into the alkaline pH forms is the same as the pK for the disappearance of the 695 nm absorption band for the cytochromes, even though these pK values range over 2 pH units. This confirms that the g1=3.06 and g1=3.2 forms contain the methionine-sulfur:heme-iron axial ligand while the g1=3.4 and the g1=3.6 forms do not. 4. At extremes of pH, the horse and bakers' yeast iso-1 proteins display several high and low spin forms that are identified, showing that a variety of protein-derived ligands will coordinate to the heme iron including methionine and cysteine sulfur, histidine imidazole, and lysine epsilon-amine. 5. The spectrum of horse cytochrome c with added azide, cyanide, hydroxide, or imidazole as axial ligands has also been examined. 6. From a comparison of the EPR and optical spectral characteristics of these groups of cytochromes with model compounds, it is suggested that the difference between them is due to a change in the hydrogen bonding or perhaps even in the protonation of N-1 of the heme iron-bound histidine imidazole.     

Fast folding of cytochrome c.

Native iso-2 cytochrome c contains two residues (His 18, Met 80) coordinated to the covalently attached heme. On unfolding of iso-2, the His 18 ligand remains coordinated to the heme iron, whereas Met 80 is displaced by a non-native heme ligand, His 33 or His 39. To test whether non-native His-heme ligation slows folding, we have constructed a double mutant protein in which the non-native ligands are replaced by asparagine and lysine, respectively (H33N,H39K iso-2). The double mutant protein, which cannot form non-native histidine-heme coordinate bonds, folds significantly faster than normal iso-2 cytochrome c: gamma = 14-26 ms for H33N,H39K iso-2 versus gamma = 200-1,100 ms for iso-2. These results with iso-2 cytochrome c strongly support the hypothesis that non-native His-heme ligation results in a kinetic barrier to fast folding of cytochrome c. Assuming that the maximum rate of a conformational search is about 10(11) s-1, the results imply that the direct folding pathway of iso-2 involves passage through on the order of 10(9) or fewer partially folded conformers.     

Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c.

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3-CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso-1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the beta-CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3-CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870-7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3-CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 beta-CH2 group and the heme 3-CH3 substituent. In addition, studies of the effect of pH on the 1H-NMR spectrum of yeast iso-1 ferricytochrome c show that the heme 3-CH3 proton resonance exhibits a pH-dependent shift with an apparent pK in the range of 6.0-7.0. The chemical shift change of the yeast iso-1 ferricytochrome c heme 3-CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934-4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C-terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3-CH3 group in yeast iso-1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8-CH3 and the 7 alpha-CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of approximately 7.0. The titrating group responsible for these spectral changes is proposed to be His39.     

Rapid intrachain binding of histidine-26 and histidine-33 to heme in unfolded ferrocytochrome C.

Time-resolved spectroscopic studies of unfolded horse iron(II) cytochrome c have suggested that the imidazole side chains of His26 and His33 bind transiently to the heme iron on microsecond time scales, after photodissociation of a carbon monoxide ligand from the heme. Our studies of four variants of cytochrome c (horse wild type, horse H33N, horse H33N/H26Q, and tuna wild type), unfolded in guanidine hydrochloride at pH 6.5, demonstrate that these side chains are responsible for the observed microsecond spectral changes. As His33 and then His26 are eliminated from the horse wild-type sequence, transient optical absorption spectra show systematic suppression of a rapid (approximately 10-100 micros) Soret absorbance change that follows photolysis of CO. Transient binding of these histidine side chains to the heme therefore generates one of the fast kinetic phases observed in previous photochemically triggered spectroscopic studies of dynamics in unfolded iron(II) cytochrome c. Furthermore, both His33 and His26 appear to contribute to a similar extent in these early kinetics. Thus, the stiffness of the polypeptide chain creates a deviation from Gaussian chain behavior by impeding, although not preventing, the formation of short (<10 peptide bonds) intrachain loops around the heme group.     

NMR comparison of prokaryotic and eukaryotic cytochromes c

1H NMR spectroscopy has been used to examine ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429) over the pH range 3.5-10.6 and the temperature range 4-60 degrees C. Resonance assignments are proposed for main-chain and side-chain protons. Comparison of results for cytochrome c-551 to recently assigned spectra for horse cytochrome c (Wand et al. (1989) Biochemistry 28, 186-194) and mutants of yeast iso-1 cytochrome (Pielak et al. (1988) Eur. J. Biochem. 177, 167-177) reveals some unique resonances with unusual chemical shifts in all cytochromes that may serve as markers for the heme region. Results for cytochrome c-551 indicate that in the smaller prokaryotic cytochrome, all benzoid side chains are rapidly flipping on the NMR time scale. In contrast, in eukaryotic cytochromes there are some rings flipping slowly on the NMR time scale. The ferrocytochrome c-551 undergoes a transition linked to pH with a pK around 7. The pH behavior of assigned resonances provides evidence that the site of protonation is the inner or buried 17-propionic acid heme substituent (IUPAC-IUB porphyrin nomenclature). Conformational heterogeneity has been observed for segments near the inner heme propionate substituent.     

Identification by proton nuclear magnetic resonance of the histidines in cytochrome b5 modified by diethyl pyrocarbonate

Diethyl pyrocarbonate (DEP) is an electrophilic reagent that is used to modify reversibly the histidine residues of proteins. Unfortunately, the lability of the acylated histidine adduct usually does not permit the isolation and identification of the modified histidine. By use of 500-MHz proton NMR spectroscopy, it has been possible to identify the C-H resonances of the nonaxial histidines of trypsin-solubilized bovine, rabbit, and porcine cytochrome b5 and therefore observe the interaction of DEP with specific histidine residues of cytochrome b5. In addition, the pKa of the peripheral histidines of bovine and rabbit cytochrome b5 have been measured in D2O. In the bovine protein it was found that the histidines are modified sequentially with increasing DEP concentration in the order His-26 greater than His-15 greater than His-80. This order is maintained in the rabbit protein with the following additions: His-26 approximately His-27 greater than His-15 greater than or equal to His-17 greater than His-80. The relative reactivity of the peripheral histidines with DEP was rationalized by considering three of their characteristics: (1) the pKa of the histidine, (2) the fraction of the side chain exposed to the solvent, and (3) the hydrogen-bond interactions of the imidazole ring.     

The identification of histidine ligands to cytochrome a in cytochrome c oxidase

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a.     

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)     

Cooperative ligand reorientations in cytochrome c3: a molecular dynamics simulation.

Molecular dynamics simulations of a tetraheme cytochrome c3 were performed to investigate dynamic aspects of the motion of the axial heme iron ligands. It was found that persistent transitions between alternate axial imidazole orientations of the histidine incorporated in the CXXCH heme binding sequence occurred via correlated motions. The correlated motions involved virtually all of the atoms comprising the polypeptide backbone of the heme binding sequence as well as the histidine imidazole side-chain.     

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.     

Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants

All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case of Rhodobacter capsulatus cytochrome c(2), the sixth heme ligand Met96 and up to 14 flanking residues (positions 88-100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region of Rhodobacter capsulatus cytochrome c(2) and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93-100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole.     

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.     

Axial histidyl imidazole non-exchangeable proton resonances as indicators of imidazole hydrogen bonding in ferric cyanide complexes of heme peroxidases

Proton NMR spectra of a model of low-spin cyanide complexes of ferric hemoproteins indicate that two broad single-protein resonances from the axial imidazole can be resolved outside the diamagnetic spectral region. Upon deprotonation of the imidazole in the model, the upfield resonance shifts dramatically to higher field, suggesting that its position may reflect the degree of hydrogen bonding or proton donation of the imidazole. Met-cyano myoglobin reveals a pair of such broad peaks in the regions expected for an essentially neutral axial imidazole. In the cyano complexes of horseradish peroxidase and cytochrome c peroxidase, a pair of single-proton resonances are located which are assigned to the same imidazole protons on the basis of their linewidth and shift changes upon altering the heme substituents. The upfiled proton, however, is found at much higher field than in metMbCN. The upfield bias of this resonance is taken as evidence for appreciable imidazolate character for the axial ligand in these heme peroxidases.     

Isolation and structure of a rat cytochrome c gene

We screened a Charon 4A-rat genomic library using the cloned iso-1 cytochrome c gene from Saccharomyces cerevisiae as a specific hybridization probe. Eight different recombinant phages homologous to a coding region subfragment of the yeast gene were isolated. Nucleotide sequence analysis of a 0.96-kilobase portion of one of these established the existence of a gene coding for a cytochrome c identical in amino acid sequence with that of mouse. The rat polypeptide chain sequence had not previously been determined. In contrast to the yeast iso-1 and iso-2 cytochrome c genes, neither of which have introns, the rat gene contains a single 105-base pair intervening sequence interrupting glycine codon 56. The overall nucleotide sequence homology between cytochrome c genes of yeast and rat is about 62%, with areas of greater homology coinciding with four regions of functionally constrained amino acid sequences. Two of these regions displayed 85-90% DNA sequence homology, including the longest consecutive homologous stretch of 14 nucleotides, corresponding to amino acids 47-52 of the rat protein. Somewhat less homology was observed in the DNA-specifying amino acids 70-80, which are invariant residues in most known cytochrome c molecules. Thermal dissociation of the yeast probe from the homologous rat DNA was at about 58 degrees C in 0.39 M Na+. These results establish that cytochrome c genes may be isolated by interspecies hybridization between widely divergent organisms.