Biomedical subjects

J G Guillemette

Publications and source records attributed to J G Guillemette.

At least 19 recordsLinked to original sources

PubMed1996

Synergy in protein engineering. Mutagenic manipulation of protein structure to simplify semisynthesis.

Semisynthesis is a chemical technique of protein engineering that provides a valuable complement to directed mutagenesis. It is the method of choice when the structural modification requires, for example, a noncoded amino acid. The process involves specific and limited protein fragmentation, structural manipulation of the target sequence, and subsequent religation of fragments to give the mutant holoprotein. We suggested and demonstrated that mutagenesis and semisynthesis could be used synergistically to achieve protein engineering goals otherwise unobtainable, if mutagenesis was used to shuffle methionine residues in the yeast cytochrome c sequence (Wallace, C. J. A., Guillemette, J. G., Hibiya, Y., and Smith, M. (1991) J. Biol. Chem. 266, 21355-21357). These residues can not only be sites of specific cleavage by CNBr but also of spontaneous peptide bond synthesis between fragments in noncovalent complexes, which greatly facilitates the semisynthetic process. We have now used an informed "methionine scan" of the protein sequence to discover other useful sites and to characterize the factors that promote this extraordinary and convenient autocatalytic religation. Of eight sites canvassed, in a wide range of settings, five efficiently provoked peptide bond synthesis. The principal factor determining efficiency seems to be the hydropathy of the religation site. The mutants created have also provided some new insights on structure-function relationships in the cytochrome.

Chemical Phenomena↗

PubMed1996

The role of a conserved water molecule in the redox-dependent thermal stability of iso-1-cytochrome c.

Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule (Wat166) at an adjacent site (Berghuis, A. M., Guillemette, J. G., McLendon, G., Sherman, F., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 236, 786-799; Berghuis, A. M., Guillemette, J. G., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 235, 1326-1341; Rafferty, S. P., Guillemette, J. G., Berghuis, A. M., Smith, M., Brayer, G. D., and Mauk, A. G. (1996) Biochemistry, 35, 10784-10792). We used differential scanning calorimetry (DSC) to determine the change in heat capacity (DeltaCp) and the temperature dependent enthalpy (DeltaHvH) for the thermal denaturation of both the oxidized and reduced forms of the iso-1 cytochrome c variants. The relative stabilities were expressed as the difference in the free energy of denaturation (DeltaGD) between the wild type and mutant proteins in both redox states. The disruption of the hydrogen bonding network results in increased stability for all of the mutant proteins in both redox states with the exception of the reduced Y67F variant which has approximately the same stability as the reduced wild type protein. For the oxidized proteins, DeltaGD values of 1.3, 4.1, 1.5, and 5.8 kcal/mol were determined for N52A, N52I, Y67F, and Y67F/N52I, respectively. The oxidized proteins were 8.2-11.5 kcal/mol less stable than the reduced proteins due to a redox-dependent increase in the entropy of unfolding.

Cytochrome c Group↗

PubMed1996

Mechanistic and structural contributions of critical surface and internal residues to cytochrome c electron transfer reactivity.

The influence of mutations in two conserved regions of yeast iso-1-cytochrome c believed to be critical to the mechanism of cytochrome c electron transfer reactions has been investigated. The variants Asn52Ala, Tyr67Phe, Ile75Met, and Thr78Gly involve perturbation of critical hydrogen-bonding interactions with an internal water molecule (Wat166) and have been studied in terms of their electrochemical properties and the kinetics with which they are reduced by Fe(EDTA)2- and oxidized by Co(phen)3(3+). In parallel studies, the Co(phen)3(3+) oxidation kinetics of Tyr, Leu, Ile, Ala, Ser, and Gly variants of the phylogenetically conserved residue Phe82 have been studied and correlated with previous electrochemical and kinetic results. To assist mechanistic interpretation of these results, the three-dimensional structures of the Asn52Ala and Ile75Met ferrocytochrome c variants have been determined. The reduction potentials of the variants modified in the region of Wat166 were at least 33 mV (pH 6, 25 degrees C, and mu = 0.1 M) lower than that of the wild-type protein. Electron transfer reactivity of this family of variants in both the oxidation and reduction reactions was increased as much as 10-fold over that of the wild-type cytochrome. On the other hand, the reactivity of the position-82 variants in both oxidation and reduction depended on the structural characteristics of the oxidation-reduction reagent with which they reacted, and this reactivity was related to the nature of the residue at this position. These findings have been interpreted as demonstrating that the principal influence of modification at position-82 arises from changes in the nature of reactant-protein interaction at the surface of the protein and in maintaining the high reduction potential of the cytochrome while the principal influence of internal modifications near Wat166 results from alteration of the reorganization energy for the oxidation state-linked conformational change defined by crystallographic analysis of the wild-type protein.

Cytochrome c Group↗

PubMed1995

Electrophoretic and spectral characterization of wild type and mutant adenovirus protease.

The P137L mutation in the adenovirus type 2 protease results in a temperature-sensitive protein-trafficking phenotype expressed during infection but not in vitro. Homology-derived secondary structure prediction placed the mutation within an externally disposed loop. Circular dichroism and urea gradient gel electrophoresis suggested that, unlike other thiol proteases, the Ad2 protease is comprised of a single conformational domain. The -0.32-kcal difference in the free energy of folding and the temperature-independent CD spectra of the mutant and wild type enzymes point to a very subtle structural change as the cause of the in vivo phenotype.

Adenoviruses, Human↗

PubMed1995

Structural studies of the roles of residues 82 and 85 at the interactive face of cytochrome c.

A combination of structural, functional, and mutagenic experiments has been used to study the roles of the invariant Phe82 and highly conserved Leu85 residues in cytochrome c, especially with respect to the complexation interface with electron transfer partners and maintenance of the hydrophobic heme pocket. Structural analyses show that the F82Y, L85A, and F82Y/L85A mutant proteins all retain the characteristic cytochrome c fold, but that conformational alterations are introduced in the direct vicinity of the mutation sites. In particular, the additional hydroxyl group of Tyr82 is in direct spatial conflict with the side chain of Leu85 in the F82Y mutant protein, leading to rotation of the side chain of Tyr82 out toward the protein surface. This strain is relieved in the F82Y/L85A mutant protein where the phenyl ring of Tyr82 is accommodated in a conformation comparable to that of the phenylalanine normally present at this location. In addition, the available space vacated by the replacement of Leu85 with an alanine allows for the inclusion of two new internal water molecules, one of which is bound to Tyr82 and the other to Arg13. In contrast, in the L85A mutant protein, no internal water molecules are observed in this exclusively hydrophobic pocket, which is partially filled by shifts in nearby side chains. Overall, the conformational changes observed result from the optimization of side chain packing to reflect the spatial requirements of new side chains, the minimization of both vacant internal space and the solvent exposure of hydrophobic groups, and the attainment of maximal hydrogen bonding between available polar groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Replacements in a conserved leucine cluster in the hydrophobic heme pocket of cytochrome c.

A cluster of highly conserved leucine side chains from residues 9, 68, 85, 94, and 98 is located in the hydrophobic heme pocket of cytochrome c. The contributions of two of these, Leu 85 and Leu 94, have been studied using a protein structure-function-mutagenesis approach to probe their roles in the maintenance of overall structural integrity and electron transfer activity. Structural studies of the L85C, L85F, L85M, and L94S mutant proteins show that, in each case, the overall fold of cytochrome c is retained, but that localized conformational shifts are required to accommodate the introduced side chains. In particular, the side chains of Cys 85 and Phe 85 form energetically favorable interactions with Phe 82, whereas Met 85 takes on a more remote conformation to prevent an unfavorable interaction with the phenyl ring of Phe 82. In the case of the L94S mutant protein, the new polar group introduced is found to form hydrogen bonds to nearby carbonyl groups. In all proteins with substitutions at Leu 85, the hydrophobic nature of the heme pocket is preserved and no significant decrease in heme reduction potential is observed. Despite earlier predictions that Leu 85 is an important determinant in cytochrome c electron transfer partner complexation, our studies show this is unlikely to be the case because the considerable surface contour perturbations made by substitutions at this residue do not correspondingly translate into significant changes in electron transfer rates. For the L94S mutant protein, the substitution of a polar hydroxyl group directly into the hydrophobic heme pocket has a larger effect on heme reduction potential, but this is mitigated by two factors. First, the side chain of Ser 94 is rotated away from the heme group and, second, the side chain of Leu 98 shifts into a portion of the new space available, partially shielding the heme group. The Leu 94 Ser substitution does not perturb the highly conserved interface formed by the nearly perpendicular packing of the N- and C-terminal helices of cytochrome c, ruling this out as the cause of this mutant protein becoming thermally labile and having a lower functional activity. Our results show these effects are most likely attributable to disruption of the heme pocket region. Much of the ability of cytochrome c to absorb the introduction of mutations at Leu 85 and Leu 94 appears to be a consequence of the conformational flexibility afforded by the leucine cluster in this region as well as the presence of a nearby internal cavity.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallography, X-Ray↗

PubMed1995

Predicted structure of the adenovirus DNA binding protein.

The DNA sequence of a portion of the MAV1 SmaI-D fragment coding for the C-terminal 147 amino acids of the adenoviral DNA-binding protein (DBP) has been determined. A multiple sequence alignment was constructed of the MAV1 fragment and the DBPs of Ad.2, 4, 5, 7, 12, 40, and 41 to examine the degree of conservation of features that have been mapped on the Ad.2 DBP and to identify further conserved features. The less conserved N-terminal segment of the protein contains two nuclear localization signals and two acidic regions, the host range region, and all of the 11 phosphorylation sites. The highly conserved C-terminal segment contains a potential leucine zipper and zinc finger motifs. These sequence features were mapped onto a predicted secondary structure of the Ad.2 DBP.

Adenoviridae↗

PubMed1994

Mutations of iso-1-cytochrome c at positions 13 and 90. Separate effects on physical and functional properties.

Residues at positions 13 (lysine or arginine) and 90 (glutamate or aspartate) of eukaryotic cytochromes c have been conserved during evolution; Cys102, however, is found only in yeast cytochrome c. The positively charged residue at position 13 and the negatively charged residue at position 90 are close together in those cytochromes c for which three-dimensional structures are available. We have replaced the amino acids at these two positions by cysteine in Saccharomyces cerevisiae iso-1-cytochrome c; in an earlier study, Cys102 was replaced by threonine without negatively influencing the physical or enzymic properties of the protein. The mutated proteins [R13C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys and Cys102-->Thr mutations), [D90C, C102T]cytochrome c (iso-1-cytochrome c containing Asp90-->Cys and Cys102-->Thr mutations) and [R13C, D90C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys, Asp90-->Cys, and Cys102-->Thr mutations) are functional in vivo. Free sulfhydryl titration shows that the doubly mutated forms each contain one sulfhydryl group while the triple mutant contains two sulfhydryl groups. The stability of mutant [R13C, C102T]cytochrome c resembles that of [C102T] cytochrome c, whereas the stability of [D90C, C102T]cytochrome c resembles the stability of [R13C, D90C, C102T]cytochrome c. The activity of cytochrome-c oxidase using cytochrome c was monitored polarographically. Compared to the wild-type or [C102T]cytochrome c, which shows two kinetic phases with cytochrome-c oxidase, [D90C, C102T]cytochrome c has much the same profile; [R13C, C102T]cytochrome c and [R13C, D90C, C102T]cytochrome c exhibit one kinetic phase with decreased activity. Electron-transfer activity of the mutant cytochromes c is inhibited by Hg2+. The inhibition is highest for the triple mutant, less for [R13C, C102T]cytochrome c, even less for [D90C, C102T]cytochrome c and insignificant for the wild type. It would appear as though the stability of the triple mutant follows the changes that result from the Asp90-->Cys mutation while the activity changes follow those of the Arg13-->Cys mutation.

Base Sequence↗

PubMed1994

The role of a conserved internal water molecule and its associated hydrogen bond network in cytochrome c.

High resolution three-dimensional structures for the N52I and N52I-Y67F yeast iso-1-cytochrome c variants have been completed in both oxidation states. The most prominent structural difference observed in both mutant proteins is the displacement of a conserved, internally bound water molecule (Wat166) from the protein matrix. In wild-type yeast iso-1-cytochrome c the position and orientation of this water molecule is found to be dependent on the oxidation state of the heme iron atom. Overall our results suggest the function of Wat166 and its associated hydrogen bond network is threefold. First, the presence of Wat166 provides a convenient mechanism to modify the hydrogen bond network involving several residues near the Met80 ligand in an oxidation state dependent manner. Second, Wat166 is necessary for the maintenance of the spatial relationships between nearby side-chains and the hydrogen bond interactions formed between these groups in this region of the protein. An essential part of this role is ensuring the proper conformation of the side-chain of Tyr67 so that it forms a hydrogen bond interaction with the heme ligand Met80. This hydrogen bond influences the electron withdrawing power of the Met80 ligand and is therefore a factor in controlling the midpoint reduction potential of cytochrome c. Elimination of this interaction in the N52I-Y67F mutant protein or elimination of Wat166 in the N52I protein with the subsequent disruption in the position and interactions of the Tyr67 side-chain, leads to a drop of approximately 56 mV in the observed midpoint reduction potential of the heme group. Third, Wat166 also appears to mediate increases in the mobility of three nearby segments of polypeptide chain when cytochrome c is in the oxidized state. Previous studies have proposed these changes may be related to oxidation state dependent interactions between cytochrome c and its redox partners. Coincident with the absence of Wat166, such mobility changes are not observed in the N52I and N52I-Y67F mutant proteins. It is possible that much of the increased protein stability observed for both mutant proteins may be due to this factor. Finally, our results show that neither heme iron charge nor heme plane distortion are responsible for oxidation state dependent conformational changes in the pyrrole A propionate region. Instead, the changes observed appear to be driven by the change in conformation that the side-chain of Asn52 experiences as the result of oxidation state dependent movement of Wat166.

Amino Acid Sequence↗

PubMed1994

Mutation of tyrosine-67 to phenylalanine in cytochrome c significantly alters the local heme environment.

The high resolution three-dimensional atomic structures of the reduced and oxidized states of the Y67F variant of yeast iso-1-cytochrome c have been completed. The conformational differences observed are localized directly in the mutation site and in the region about the pyrrole A propionate. Shifts in atomic positions are largely restricted to nearby amino acid side-chains, whereas little perturbation of the polypeptide chain backbone is observed. One prominent difference between the variant and wild-type structures involves a substantial increase in the size of an already existing internal cavity adjacent to residue 67. This same cavity contains an internally bound water molecule (Wat166), which is found in all eukaryotic cytochromes c for which structures are available. In the reduced Y67F mutant protein a second water molecule (Wat300) is observed to reside in this enlarged internal cavity, assuming a position approximately equivalent to that of the hydroxyl group of Tyr67 in the wild-type protein. A further consequence of this mutation is the alteration of the hydrogen bond network between Tyr67, Wat166 and other nearby residues. This appears to be responsible for the absence of oxidation state dependent changes in polypeptide chain flexibility observed in the wild-type protein. Furthermore, loss of the normally resident Tyr67 OH to Met80 SD hydrogen bond leads to a significantly lower midpoint reduction potential. These results reaffirm proposals that both Tyr67 and Wat166 play a central role in stabilizing the alternative oxidation states of cytochrome c.

Cytochrome c Group↗

PubMed1994

Analysis of the bimolecular reduction of ferricytochrome c by ferrocytochrome b5 through mutagenesis and molecular modelling.

Site-directed mutagenesis has been used to produce variants of cytochrome c in which selected structural or functional properties of this protein are altered that have been implicated previously in contributing to the rate at which ferricytochrome c is reduced by ferrocytochrome b5. In total, 18 variants have been studied by kinetics and electrochemical methods to assess the contributions of thermodynamic driving force, surface charge and hydrophobic interactions, and redox-linked structural reorganization of the protein to the rate of electron transfer between these two proteins under conditions where the reaction is bimolecular. While some variants (those at position-38) appear to affect primarily the driving force of the reaction, others appear to influence the rearrangement barrier to electron transfer (those at positions-67 and -52) while the interface between electron donor and acceptor centers is the principal effect of substitutions for a conserved aromatic heme contact residue at the surface of the protein (position-82). Interpretation of these results has been facilitated through the use of energy minimization calculations to refine the hypothetical models previously suggested for the cytochrome c- cytochrome b5 precursor complex on the basis of Brownian dynamics simulations of the bimolecular encounter event.

Amino Acid Sequence↗

PubMed1993

Effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso-1-ferricytochrome c by bovine ferrocytochrome b5.

The reduction of wild-type yeast iso-1-ferricytochrome c (ycytc) and several mutants by trypsin-solubilized bovine liver ferrocytochrome b5 (cytb5) has been studied under conditions in which the electron-transfer reaction is bimolecular. The effect of electrostatic charge modifications and steric changes on the kinetics has been determined by experimental and theoretical observations of the electron-transfer rates of ycytc mutants K79A, K'72A, K79A/K'72A, and R38A (K' is used to signify trimethyllysine (Tml)). A structurally robust Brownian dynamics (BD) method simulating diffusional docking and electron transfer was employed to predict the mutation effect on the rate constants. A realistic model of the electron-transfer event embodied in an intrinsic unimolecular rate constant is used which varies exponentially with donor-acceptor distance. The BD method quantitatively predicts rate constants over a considerable range of ionic strengths. Semiquantitative agreement is obtained in predicting the perturbing influence of the mutations on the rate constants. Both the experimentally observed rate constants and those predicted by BD descend in the following order: native ycytc > K79A > K'72A > K79A/K'72A. Variant R38A was studied at a different ionic strength than this series of mutations, and the theory agreed with experiment in predicting a smaller rate constant for the mutant. In all cases the predicted effect of mutation was in the correct direction, but not as large as that observed. The BD simulations predict that the two proteins dock through essentially a single domain, with a distance of closest approach of the two heme groups in rigid body docking typically around 12 A. Two predominant classes of complexes were calculated, the most frequent involving the quartet of cytb5/ycytc interactions, Glu48-Arg13, Glu56-Lys87, Asp60-Lys86, and heme-Tml72, having an average electrostatic energy of -13.0 kcal/mol. The second most important complexes were of the type previously postulated (Salemme, 1976; Mauk et al., 1986; Rodgers et al., 1988) with interactions Glu44-Lys27, Glu48-Arg13, Asp60-Tml72, and heme-Lys79 and having an energy of -6.4 kcal/mol. The ionic strength dependence of the bimolecular reaction rate was well reproduced using a discontinuous dielectric model, but poorly so for a uniform dielectric model.

Amino Acid Sequence↗

PubMed1993

Redesign of the interior hydrophilic region of mitochondrial cytochrome c by site-directed mutagenesis.

Heme propionate-7 in cytochrome c is an ionizable group located in a region of the protein that is inaccessible to bulk solvent. Electrostatic stabilization of this functional group appears to be achieved through interaction of heme propionate-7 with several amino acid residues that occur within hydrogen-bonding distance of it. To investigate the functional and spectroscopic roles of the amino acid residues that contribute to the immediate environment of heme propionate-7, the following variant forms of yeast (Saccharomyces cerevisiae) cytochrome c have been prepared and characterized by electrochemical and spectrochemical analyses: Arg38Ala, Tyr48Phe, Ala38Phe, Tyr48Phe/Trp59Phe, and Arg38Ala/Tyr48Phe/Trp59Phe. For each protein, the dependence of midpoint reduction potential and NMR spectrum on pH was determined, and the UV (250-450 nm) circular dichroic (CD) spectrum was measured. All of the variant proteins exhibited decreased reduction potentials with the greatest difference (-65 to -70 mV) exhibited by the multiply mutated proteins. The electrostatic properties of the variant proteins as reflected by the oxidation-state dependence of the His-39 pKa value were similar to those of the wild-type protein. Previous indirect assignments of minima in the CD spectrum of cytochrome c at 282 and 289 nm to Trp-59 are confirmed by spectra of the variant cytochromes in which this residue is replaced by Phe. The present results establish that the electrochemical effects of eliminating hydrogen-bonding interactions with heme propionate-7 are not additive and that the functional modulation of cytochrome c through regulation of the heme propionate-7 dielectric environment involves a complex combination of solvation effects and electrostatic or hydrogen-bonding interactions.

Base Sequence↗

PubMed1991

NMR study of the structural characteristics of variants of yeast iso-1-cytochrome c in which unvaried aromatic residues have been substituted.

The structures of variants of yeast iso-1-cytochrome c, in which the previously unchanged Tyr48 and Tyr48 + Trp59 have been replaced by Phe, have been characterised by NMR. The NMR data indicated that the structures of the variant cytochromes c are very similar to the wild-type protein. In particular, the heme environment and interactions of the heme macrocycle were shown to be preserved. The observation of chemical shift differences have allowed for the assessment of conformational changes. The substitution of Trp59 by Phe may have caused a small conformational change, a manifestation of which is the observed chemical shift differences at His39, Val57 and Tyr74. The structural basis for the reduction in redox potential accompanying the amino acid substitutions is discussed and the proposal made that the changes in potential are a direct consequence of the side chain properties and do not result primarily from conformational changes.

Amino Acids↗

PubMed1991

Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis.

If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively, bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation, for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic ones, that are appropriate to the semisynthetic goals.

Amino Acid Sequence↗

PubMed1991

Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector.

A phagemid (pING4) carrying the yeast iso-1-cytochrome c gene was constructed which bears all the elements necessary for replication in yeast and bacteria and may be converted into a single-stranded form of DNA for site-directed mutagenesis and nucleotide sequencing. The recombinant vector was used to create a complete set of 19 amino acid changes at position 82, a phylogenetically conserved phenylalanine residue in mitochondrial cytochrome c. All the different forms of cytochrome c were functional in vivo, based upon their ability to support respiration when the mutant proteins were expressed in a yeast strain (otherwise devoid of cytochrome c) grown on non-fermentable carbon sources, with only the strain containing the Cys82 variant having a substantially decreased growth rate. These results are interpreted in terms of the available structural and functional information previously reported on a subset of cytochrome c proteins with mutations at position 82.

Amino Acid Sequence↗

PubMed1991

Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin.

A gene for expression of horse heart myoglobin in Escherichia coli has been constructed in one step from long synthetic oligonucleotides. The synthetic gene contains an efficient translation initiation signal and used codons that are commonly found in E. coli. Unique restriction sites are placed throughout the gene. It has been inserted in a phagemid vector and is expressed from the lac promoter in E. coli at high efficiency, the soluble heme protein representing approximately 10% of soluble protein. Two versions of horse heart myoglobin were produced with aspartic acid or asparagine at residue 122. Comparison of chromatographic mobilities of these two proteins with authentic horse heart myoglobin identified aspartic acid as the correct residue 122. The availability of this gene, which is designed to facilitate oligonucleotide mutagenesis or cassette mutagenesis, will allow systematic structure-function analysis of horse heart myoglobin.

Amino Acid Sequence↗

PubMed1990

Electrochemical, kinetic, and circular dichroic consequences of mutations at position 82 of yeast iso-1-cytochrome c.

Replacement of Phe-82 in yeast iso-1-cytochrome c with Tyr, Leu, Ile, Ser, Ala, and Gly produces a gradation of effects on (1) the reduction potential of the protein, (2) the rate of reaction with Fe(EDTA)2-, and (3) the CD spectra of the ferricytochromes in the Soret region under conditions where contributions from the alkaline forms of these proteins are absent. The reduction potential of cytochrome c is lowered by as little as 10 mV (Tyr-82) or by as much as 43 mV (Gly-82; pH 6.0) as the result of these substitutions. The second-order rate constants for reduction of these cytochromes range from a low of 6.20 (2) x 10(4) for the Tyr-82 variant to a high of 14.8 x 10(4) M-1 s-1 for the Ser-82 variant [pH 6.0, 25 degrees C, mu = 0.1 M (sodium phosphate)]. Analysis of these rates by use of relative Marcus theory produces values of k11corr that range from 10.9 M-1 s-1 for the wild-type protein to 190 M-1 s-1 for the Gly-82 mutant [25 degrees C, mu = 0.1 M, pH 6.0 (sodium phosphate)]. Reinvestigation of the effect of substituting Phe-82 by a Tyr residue on the CD spectrum of the protein now reveals little alteration of the intense, negative Cotton effect in the Soret CD spectrum of ferricytochrome c. On the other hand, substitution of nonaromatic residues of various sizes at this position results in loss of this spectroscopic feature, consistent with previous findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗