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.     

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.     

Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c

Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5-13C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 degrees C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in slow exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters: delta H++ = 28 kcal/mol for both I and III; delta S++ = 42 cal/(mol.K) for I, and delta S++ = 41 cal/(mol.K) for III. The remaining tyrosine (V) has a larger enthalpy and entropy of activation: delta H++ - 36 kcal/mol, delta S++ = 72 cal/(mol.K). Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

pH dependence of folding of iso-2-cytochrome c

Starting from a standard unfolded state (3.0 M guanidine hydrochloride, pH 7.2), the kinetics of refolding of iso-2-cytochrome c have been investigated as a function of final pH between pH 3 and pH 10. Absorbance in the ultraviolet and visible spectral regions and tryptophan fluorescence are used to monitor folding. Over most of the pH range, fast and slow folding phases are detected by both fluorescence and absorbance probes. Near neutral pH, the rate of fast folding appears to be the same when monitored by absorbance and fluorescence probes. At higher and lower pH, there are two fast folding reactions, with absorbance-detected fast folding occurring in a slightly faster time range than fluorescence-detected fast folding. The rates of both fast folding reactions pass through broad minima near neutral pH, indicating involvement of ionizable groups in rate-limiting steps. The rates of slow folding also depend on the final pH. At acid pH, there appears to be a single slow folding phase for both fluorescence and absorbance probes. At neutral pH, the absorbance-detected and fluorescence-detected slow folding phases separate into distinct kinetic processes which differ in rate and relative amplitude. At high pH, absorbance-detected slow folding is no longer observed, while fluorescence-detected slow folding is decreased in amplitude. In contrast, the equilibrium and kinetic properties of proline imide bond isomerization, believed to be involved in the slow folding reactions, are largely independent of pH. The results suggest that the pH dependence of slow folding involves coupling of pH-sensitive structure to proline imide bond isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Yeast mutants defective in iso-2-cytochrome c.

The structural gene CYC7 for yeast iso-2-cytochrome c was previously identified by isolating a mutant, cyc7-1-1, totally lacking iso-2-cytochrome c and demonstrating that revertants of this mutant contained iso-2-cytochrome c with an altered primary structure (Downie et al., 1977). In this paper we describe a variety of different types of mutants that completely or partially lack iso-2-cytochrome c due to mutations in either the structural gene, CYC7, or unlinked “regulatory” genes. The iso-2-cytochrome c-deficient mutants were isolated by benzidine staining of over 3 × 10⁵ colonies from ?^? strains (cytoplasmic petites) that lacked iso-1-cytochrome c due to the deletion cyc1-1 and that contain abnormally high levels of iso-2-cytochrome c due to a chromosomal translocation, CYC7-1, adjacent to the normal structural gene CYC7 +. The cytochrome c content of mutants not staining with the benzidine reagents was estimated by low temperature spectroscopy, and 139 mutants containing significantly decreased levels of iso-2-cytochrome c were analyzed genetically by complementation with previously identified cyc mutants. In this way 50 mutants at the cyc2 and cyc3 loci were identified along with a group of 62 mutants of the structural gene cyc7. The different types of mutants of the structural gene which were uncovered and which were more or less anticipated included those that completely lacked iso-2-cytochrome c, those that were suppressible by UAA or UAG suppressors, those that lacked iso-2-cytochrome c but had increased levels after growth at lower temperatures, and those that exhibited visibly altered c_a absorption bands of iso-2-cytochrome c. Iso-2-cytochrome c mutants with altered primary structures were obtained from intragenic revertants of several of these mutants, confirming our earlier conclusion that cyc7 is the structural gene. In addition we observed an unexpected class of mutants that lacked iso-2-cytochrome c when in the ?^? state but contained approximately the CYC7-1 parental level when in the ?⁺ state. Two of these mutants, cyc7-1-47 and cyc7-1-49, were shown to contain altered iso-2-cytochromes c. The different contents of the abnormal iso-2cytochromes c suggest that cytochrome c has different environments in ?⁺ and ?^? mitochondria and that the ?⁺ condition may stabilize certain altered proteins.     

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.     

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)     

Demonstration of several independent loci involved in the synthesis of iso-2-cytochrome c in yeast

Summary This study concerns the chromosomal genes controlling the synthesis of cytochrome c in yeast. In the wild type there are two molecular species of cytochrome c : iso-1 (major from) and iso-2 (minor form) which differ in many positions of their amino-acid sequence. A mutation, CY1cy1-1, in the structural gene for iso-1, leads to iso-1 deficiency, while retaining a normal albeit small amount of iso-2-cytochrome c.The cyI-1 mutant does not grow on DL-lactate as sole carbon source, while the wild type does. This property was used for selecting cytochrome c rich revertants (CYT) from cytochrome c deficient strains cy1-1; ca 200 revertants were isolated after extensive nitrous acid mutagenesis from a haploid cy1-1 strain or from a diploid cy1-1/cy1-1 strain and ca 30 of them were analyzed genetically and biochemically. The cytochrome c of seven (CYT) revertants was extracted and characterized; none of them contained iso-1-cytochrome c, but all contained large amount of iso-2-cytochrome csufficient to compensate for the deficiency. It was concluded that none of the revertants resulted from back mutation of cy1-1 and that the cy1-1 mutation is a deletion or some other irreversible aberration. These conclusions were corroborated by genetic analysis. It was shown that every reversion is due to a chromosomal mutation segregating as a single gene. Five unlinked gene loci, CY2A, CY2B, CY2C, CY2D, CY2E, were uncovered in this way. None of them were linked to the CY1 locus. Revertants selected in the diploid strain were dominant or semi-dominant while those selected in the haploid strain were recessive. To the first class belong alleles at loci CY2A, CY2B, CY2C, while to the latter belong alleles at loci CY2D and CY2E.Five unlinked loci are implicated in iso-2-cytochrome c synthesis. Mutations selected at these loci act as suppressors of cytochrome c deficiency caused by a deletion of the CY1 locus. In fact the muations do not restore the synthesis of the deficient protein (iso-1-cytochrome c), but increase the synthesis of an another protein, structurally alike (iso-2-cytochrome c), and having very similar if not identical physiological activity. We propose the term of compensator genes to define this type of mutations. We discuss some possible mechanisms to explain the rarity of compensator mutations and the hypothesis that the locus CY2A could correspond not only to the regulatory gene for iso-2-cytochrome c but also to the structural one.     

Structure determination and analysis of yeast iso-2-cytochrome c and a composite mutant protein.

As part of a study of protein folding and stability, the three-dimensional structures of yeast iso-2-cytochrome c and a composite protein (B-2036) composed of primary sequences of both iso-1 and iso-2-cytochromes c have been solved to 1.9 A and 1.95 A resolutions, respectively, using X-ray diffraction techniques. The sequences of iso-1 and iso-2-cytochrome c share approximately 84% identity and the B-2036 composite protein has residues 15 to 63 from iso-2-cytochrome c with the rest being derived form the iso-1 protein. Comparison of these structures reveals that amino acid substitutions result in alterations in the details of intramolecular interactions. Specifically, the substitution Leu98Met results in the filling of an internal cavity present in iso-1-cytochrome c. Further substitutions of Val20Ile and Cys102Ala alter the packing of secondary structure elements in the iso-2 protein. Blending the isozymic amino acid sequences in this latter area results in the expansion of the volume of an internal cavity in the B-2036 structure to relieve a steric clash between Ile20 and Cys102. Modification of hydrogen bonding and protein packing without disrupting the protein fold is illustrated by the His26Asn and Asn63Ser substitutions between iso-1 and iso-2-cytochromes c. Alternatively, a change in main-chain fold is observed at Gly37 apparently due to a remote amino acid substitution. Further structural changes occur at Phe82 and the amino terminus where a four residue extension is present in yeast iso-2-cytochrome c. An additional comparison with all other eukaryotic cytochrome c structures determined to date is presented, along with an analysis of conserved water molecules. Also determined are the midpoint reduction potentials of iso-2 and B-2036 cytochromes c using direct electrochemistry. The values obtained are 286 and 288 mV, respectively, indicating that the amino acid substitutions present have had only a small impact on the heme reduction potential in comparison to iso-1-cytochrome c, which has a reduction potential of 290 mV.     

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.     

Network of interactions between unlinked genes: synergistic and antagonistic regulation of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 synthesis]

Five chromosomal genes, CYPI to CYP5 involved in the regulation of the synthesis of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 are described. The function of these genes was studied either by varying the proportion of the mutated and wild type alleles in the cell vy varing the growth conditions, or else by transforming the mutants into sigma-cytoplasmic petites. We have shown a network of genetic interactions which regulate the synthesis of three structurally different proteins : iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2, by two unlinked genes : CYC1 and CYP1, one of which (CYC1) is the structural gene by iso-1-cytochrome c. Within this network the interactions are proportional to the gene dosage and are either antagonistic or synergistic depending on the allele combination and the protein studied. The mutated alleles cyp1 stimulate the synthesis of iso-2-cytochrome c, inhibit the synthesis of iso-1-cytochrome c, while the cytochrome b2 synthesis is also inhibited but by a combination of cyp1 mutated alleles CYC1 wild type allele. Other loci, CYP2, CYP3, CYP4 and CYP5 were also studied in various allelic combinations. They show some interactions between them or with CYC1 locus but these interactions are different and less pronounced than those involving loci CYP1 and CYC1.     

Free energy of transition for the individual alkaline conformers of yeast iso-1-cytochrome c

Direct protein electrochemistry was used to obtain the thermodynamic parameters of transition from the native (state III) to the alkaline (state IV) conformer for untrimethylated Saccharomyces cerevisiae iso-1-cytochrome c expressed in E. coli and its single and multiple lysine-depleted variants. In these variants, one or more of the lysine residues involved in axial Met substitution (Lys72, Lys73, and Lys79) was mutated to alanine. The aim of this work is to determine the thermodynamic affinity of each of the substituting lysines for the heme iron and evaluate the interplay of enthalpic and entropic factors. The equilibrium constants for the deprotonation reaction of Lys72, 73, and 79 were computed for the minimized MD average structures of the wild-type and mutated proteins, applying a modified Tanford-Kirkwood calculation. Solvent accessibility calculations for the substituting lysines in all variants were also performed. The transition enthalpy and entropy values within the protein series show a compensatory behavior, typical of a process involving extensive solvent reorganization effects. The experimental and theoretical data indicate that Lys72 most readily deprotonates and replaces M80 as the axial heme iron ligand, whereas Lys73 and Lys79 show comparably higher pKa values and larger transition free energies. A good correlation is found within the series between the lowest calculated Lys pKa value and the corresponding experimental pKa value, which can be interpreted as indicative of the deprotonating lysine itself acting as the triggering group for the conformational transition. The triple Lys to Ala mutant, in which no lysine residues are available for heme iron binding, features transition thermodynamics consistent with a hydroxide ion replacing the axial methionine ligand.