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)     

Changes in conformation and slow refolding kinetics in mutant iso-2-cytochrome c with replacement of a conserved proline residue

Using oligonucleotide-directed mutagenesis, we have produced a mutant form of iso-2-cytochrome c of yeast in which threonine (Thr-71) replaces a conserved proline residue (Pro-71) located between two short alpha-helical segments in the native protein. Optical spectroscopy indicates that, at pH 7.2, Thr-71 iso-2-cytochrome c folds to a nonnative conformation possibly related to the alkaline form of the native protein. On titration to pH 5.2, Thr-71 iso-2-cytochrome c regains many of the optical properties of the normal protein. We have shown that the proline residue at position 71 has no effect on the kinetics of fluorescence-detected slow refolding. However, between pH 5 and pH 7.2 the amplitude for absorbance-detected slow folding is strongly pH dependent in the mutant protein but is largely independent of pH in the normal protein. We believe this to be due to the folding of Thr-71 iso-2-cytochrome c to a nonnative conformation at pH 7.2 that does not require the slow, absorbance-detected conformational changes observed in folding to the more native-like state at pH 5-6.     

Substitutions of proline 76 in yeast iso-1-cytochrome c. Analysis of residues compatible and incompatible with folding requirements

Fine-structure genetic mapping previously revealed numerous nonfunctional cyc1 mutations having alterations at or near the site corresponding to amino acid position 76 of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. DNA sequencing of the alterations in four of these cyc1 mutations indicated that the normal Pro-76 was replaced by Leu-76. Revertants containing at least partially functional iso-1-cytochromes c were isolated, and the alterations were analyzed by DNA sequencing and protein analysis. Specific activities of the altered iso-1-cytochromes c were estimated in vivo by growth of the strains in lactate medium; compared to normal iso-1-cytochrome c with Pro-76, the following activities were associated with the following replacements: approximately 90% for Val-76, approximately 60% for Thr-76, approximately 30% for Ser-76, approximately 20% for Ile-76, and 0% for Leu-76. In order to develop an understanding of the factors that determine whether or not an altered iso-1-cytochrome c will function, we undertook a theoretical analysis which led to the conclusion that the activity of the proteins was dependent on both short- and long-range interactions. Short-range interactions were estimated from studies on known protein structures which gave the likelihood that various amino acids would be found in a local backbone configuration similar to the native protein; long-range interactions with the rest of the molecule were analyzed by considering the size of the side chain. We believe this approach can be used to analyze a wide variety of mutant proteins.     

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.     

Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c

As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine [Wood, L. C., Muthukrishnan, K., White, T. B., Ramdas, L., & Nall, B. T. (1988) Biochemistry (preceding paper in this issue)]. For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 degrees C (delta G degrees u = 3.8 kcal/mol for normal Pro-76 iso-2 versus 2.6 kcal/mol for Gly-76 iso-2). The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing. The rates for fast and slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2. A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfolding of the mutant protein. The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species. The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manual mixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanidine hydrochloride induced equilibrium unfolding of mutant forms of iso-1-cytochrome c with replacement of proline-71

Proline-71, an evolutionally conserved residue that separates two short alpha-helical regions, is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, J. F., Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 13225-13236]. Treatment of these proteins with a specific sulfhydryl blocking reagent (methyl methanethiosulfonate) to block Cys-102 has allowed investigation of the properties of monomeric forms of the proteins, denoted iso-1-MS. Comparison of the UV-visible absorbance properties (pH 6, 20 degrees C) shows minor differences between the normal Pro-71 iso-1-MS and two of the three mutant proteins. The Val-71 iso-1-MS protein has absorbance properties indistinguishable from those of the normal Pro-71 iso-1-MS protein, but the Ile-71 iso-1-MS and Thr-71 iso-1-MS proteins show reduced intensity of the 695-nm absorbance band and a small shift in the Soret maximum, from 408 nm for the Pro-71 iso-1-MS and Val-71 iso-1-MS proteins to 406 nm for the Thr-71 iso-1-MS and Ile-71 iso-1-MS proteins. Second derivative spectroscopy is used to assess differences in the polarity of the environment of tyrosine residues. The average degree of exposure of tyrosines to solvent is similar in all four proteins: 0.39 for the normal Pro-71 iso-1-MS and Val-71 iso-1-MS proteins; 0.40 for the Ile-71 iso-1-MS protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Probing the dynamics of a His73-heme alkaline transition in a destabilized variant of yeast iso-1-cytochrome c with conformationally gated electron transfer methods

The alkaline transition of cytochrome c involves substitution of the Met80 heme ligand of the native state with a lysine ligand from a surface Ω-loop (residues 70 to 85). The standard mechanism for the alkaline transition involves a rapid deprotonation equilibrium followed by the conformational change. However, recent work implicates multiple ionization equilibria and stable intermediates. In previous work, we showed that the kinetics of formation of a His73-heme alkaline conformer of yeast iso-1-cytochrome c requires ionization of the histidine ligand (pK(HL) ~ 6.5). Furthermore, the forward and backward rate constants, k(f) and k(b), respectively, for the conformational change are modulated by two auxiliary ionizations (pK(H1) ~ 5.5, and pK(H2) ~ 9). A possible candidate for pK(H1) is His26, which has a strongly shifted pK(a) in native cytochrome c. Here, we use the AcH73 iso-1-cytochrome c variant, which contains an H26N mutation, to test this hypothesis. pH jump experiments on the AcH73 variant show no change in k(obs) for the His73-heme alkaline transition from pH 5 to 8, suggesting that pK(H1) has disappeared. However, direct measurement of k(f) and k(b) using conformationally gated electron transfer methods shows that the pH independence of k(obs) results from coincidental compensation between the decrease in k(b) due to pK(H1) and the increase in k(f) due to pK(HL). Thus, His26 is not the source of pK(H1). The data also show that the H26N mutation enhances the dynamics of this conformational transition from pH 5 to 10, likely as a result of destabilization of the protein.     

Mutation-induced perturbation of the cytochrome c alkaline transition

The possible influence of residue Phe-82 in the cytochrome c alkaline isomerization has been evaluated by spectrophotometric pH titrations of a family of mutant yeast iso-1-cytochromes c in which the identity of the residue at this position has been varied. The pKa for the exchange of the Met-80 heme iron ligand was determined from pH titrations in which the S----Fe charge-transfer band (695 nm) was monitored and was found to be 8.5 for the wild type, 7.7 for Ser-82, 7.7 for Gly-82, 7.2 for Leu-82, and 7.2 for Ile-82. pH-jump experiments [Davis et al. (1974) J. Biol. Chem. 249, 2624] established that substitutions at position 82 affect the alkaline isomerization by lowering the pKa of the titrating group by as much as 1.4 pK units; for the Ser-82 and Gly-82 variants, there is also a small effect on the Keq for the ligand exchange equilibrium. On the basis of these findings, we conclude that one critical role for Phe-82 in the wild-type protein is stabilization of the native heme binding environment.     

Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c

Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, J. J., Jr., Muthukrishnan, K., & Nall, B. T. (1985) Biochemistry 24, 6680-6684]. The kinetics of absorbance-detected refolding of the alkaline form have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (tau 2) are detected, as in refolding to the native state at neutral pH. An additional kinetic phase (tau a) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparison to pH-jump measurements of the fully folded protein shows that phase tau a has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species--with a 695-nm absorbance band--are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus, (1) refolding to the alkaline form of iso-2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leading to native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkaline isomerization of horse and yeast cytochromes C. Spectrophotometric and circular dichroism studies.

Spectrophotometric studies of the alkaline isomerization of horse heart and yeast cytochrome c show that the haemoproteins from Saccharomyces cerevisiae differ significantly from the mammalian cytochrome c. Apparent pKa values of 8.41, 8.40 and 8.73 for isol-1-(the methylated and unmethylated forms) and iso-2-cytochrome c respectively, from baker's yeast were determined and compared with the value of 9.40 found for horse heart cytochrome c. The transitions, measured by observing the decrease of the absorbance at 695 nm as the pH increases, have been found to strictly parallel the decrease in amplitude of the negative circular dichroism band centered at 417 nm. This observation gives additional evidence that this negative band is closely related to the ligation of the heme iron by the sulfur atom of methionine 8u for each of the four haemoproteins examined.     

Compressing the free energy range of substructure stabilities in iso‐1‐cytochrome c

Evolutionary conservation of substructure architecture between yeast iso-1-cytochrome c and the well-characterized horse cytochrome c is studied with limited proteolysis, the alkaline conformational transition and global unfolding with guanidine-HCl. Mass spectral analysis of limited proteolysis cleavage products for iso-1-cytochrome c show that its least stable substructure is the same as horse cytochrome c. The limited proteolysis data yield a free energy of 3.8 ± 0.4 kcal mol⁻¹ to unfold the least stable substructure compared with 5.05 ± 0.30 kcal mol⁻¹ for global unfolding of iso-1-cytochrome c. Thus, substructure stabilities of iso-1-cytochrome c span only ∼1.2 kcal mol⁻¹ compared with ∼8 kcal mol⁻¹ for horse cytochrome c. Consistent with the less cooperative folding thus expected for the horse protein, the guanidine-HCl m-values are ∼3 kcal mol⁻¹M⁻¹ versus ∼4.5 kcal mol⁻¹M⁻¹ for horse versus yeast cytochrome c. The tight free energy spacing of the yeast cytochrome c substructures suggests that its folding has more branch points than for horse cytochrome c. Studies on a variant of iso-1-cytochrome c with an H26N mutation indicate that the least and most stable substructures unfold sequentially and the two least stable substructures unfold independently as for horse cytochrome c. Thus, important aspects of the substructure architecture of horse cytochrome c, albeit compressed energetically, are preserved evolutionally in yeast iso-1-cytochrome c.     

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.     

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.     

Role of phenylalanine-82 in yeast iso-1-cytochrome c and remote conformational changes induced by a serine residue at this position

A three-dimensional structural analysis of the reduced form of the Ser-82 mutant protein of yeast iso-1-cytochrome c has been completed to 2.8-A resolution. Replacement of Phe-82 with a serine residue results in conformational changes both near and remote from the mutation site. Those groups undergoing positional shifts near Ser-82 include Arg-13, Gly-83 and -84, and the CBB methyl of the heme group. Remote shifts are centered about the propionate of pyrrole ring A and principally involve Asn-52, Trp-59, and an internally buried water molecule, WAT-166. Placement of a serine side chain at position 82 also leads to the formation of a large solvent channel which substantially increases the solvent accessibility of the heme group. This would appear to account for the much lower reduction potential observed for this protein. The detrimental effect of Ser-82 on both the steady-state activity and the rate of electron transfer in complexation with cytochrome c peroxidase can also be interpreted in terms of the modified character of the region about the mutation site. The remote conformational changes observed appear to represent the equivalent of the initial conformational changes occurring as yeast iso-1-cytochrome c is converted to the fully oxidized state during an electron-transfer event. These results agree well with the proposal [Moore, G. R. (1983) FEBS Lett. 161, 171-175] that the trigger for conformational changes between oxidation states resides in the nature of the interactions between the heme iron atom and the pyrrole ring A propionate group.     

pH dependence of formation of a partially unfolded state of a Lys 73 --> His variant of iso-1-cytochrome c: implications for the alkaline conformational transition of cytochrome c

The alkaline conformational transition of a lysine 73 --> histidine variant of iso-1-cytochrome c has been studied. The transition has been monitored at 695 nm, a band sensitive to the presence of the heme-methionine 80 bond, at the heme Soret band which is sensitive to the nature of the heme ligand, and by NMR methods. The guanidine hydrochloride dependence of the alkaline conformational transition has also been monitored. The histidine 73 protein has an unusual biphasic alkaline conformational transition at both 695 nm and the heme Soret band, consistent with a three-state process. The conformational transition is fully reversible. An equilibrium model has been developed to account for this behavior. With this model, it has been possible to obtain the acid constant for the trigger group, pK(H), of the low-pH phase from the equilibrium data. A pK(H) value of 6.6 +/- 0.1 in H(2)O was obtained, consistent with a histidine acting as the trigger group. The NMR data for the low-pH phase of the alkaline conformational transition are consistent with an imidazole ligand replacing Met 80. For the high-pH phase of the biphasic alkaline transition, the NMR data are consistent with lysine 79 being the heme ligand. Guanidine hydrochloride m values of 1.67 +/- 0.08 and 1.1 +/- 0.2 kcal mol(-1) M(-1) were obtained for the low- and high-pH phases of the biphasic alkaline transition of the histidine 73 protein, respectively, consistent with a greater structural disruption for the low-pH phase of the transition.     

Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant.

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.     

Replacement of cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c with threonine: improved stability of the mutant protein

Site-directed mutagenesis has been used to change the codon for cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c to a threonine codon. The resulting protein is active in vivo, is methylated as in the wild-type protein and has optical properties indistinguishable from those of the wild-type protein. The threonine-107 iso-1-cytochrome c demonstrated fully reversible electrochemical behaviour and a mid-point reduction potential of 272 mV versus NHE. In addition, this mutant does not demonstrate a tendency to autoreduce or to dimerize as does the wild-type protein. These properties of the threonine-107 mutant establish that it will provide a useful background in which to make subsequent mutations for mechanistic and physical studies of yeast iso-1-cytochrome c.     

Genes Affecting the Expression of Cytochrome c in Yeast: Genetic Mapping and Genetic Interactions

The four mutant genes, cyc2, cyc3, cyc8 and cyc9, that affect the levels of the two iso-cytochromes c in the yeast Saccharomyces cerevisiae have been characterized and mapped. Both cyc2 and cyc3 lower the amount of iso-1-cytochrome c and iso-2-cytochrome c; whereas, cyc8 and cyc9 increase the amount of iso-2-cytochrome c. The cyc2, cyc3, cyc8 and cyc9 genes are located, respectively, on chromosomes XV, I, II and III, and are, therefore, unlinked to each other and unlinked to CYC1, the structural gene of iso-1-cytochrome c and to CYC7, the structural gene of iso-2-cytochrome c. While some cyc3 mutants are completely or almost completely deficient in cyotchromes c, none of the cyc2 mutants contained less than 10% of parental level of cytochrome c even though over one-half of the mutants contain UAA or UAG nonsense mutations. Thus, it appears as if a complete block of the cyc2 gene product still allows the formation of a residual fraction of cytochrome c. The cyc2 and cyc3 mutant genes cause deficiencies even in the presence of CYC7, cyc8 and cyc9, which normally cause overproduction of iso-2-cytochrome c. We suggest that cyc2 and cyc3 may be involved with the regulation or maturation of the iso-cytochromes c. In addition to having high levels of iso-2-cytochromes c, the cyc8 and cyc9 mutants are associated with flocculent cells and other abnormal phenotypes. The cyc9 mutant was shown to be allelic with the tup1 mutant and to share its properties, which include the ability to utilize exogenous dTMP, a characteristic flocculent morphology, the lack of sporulation of homozygous diploids and low frequency of mating and abnormally shaped cells of alpha strains. The diverse abnormalities suggest that cyc8 and cyc9 are not simple regulatory mutants controlling iso-2-cytochrome c.