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The Thr-102 mutant of yeast iso-1-cytochrome c is a useful system for the study of structure-function relationships in this important class of electron transfer proteins, but little is known about its structure. Furthermore, few assignments of individual amino acid residues in yeast iso-1-cytochrome c have been made by proton NMR. Here we report assignments for nearly half of the amino acids in the reduced Thr-102 mutant of yeast iso-1-cytochrome c. We also report assignments for the oxidized Thr-102 mutant. While the crystal structure of the reduced iso-1-cytochrome c (N. B. not the Thr-102 mutant) has been reported, there is currently little structural information concerning its solution structure and none concerning the oxidized protein. There is also no information concerning the structure of either oxidation state of the Thr-102 mutant. Comparison of the chemical shift and NOE data for the reduced Thr-102 mutant and comparison of paramagnetic shifts for analogous residues between this mutant and horse-heart and tuna cytochromes c reveal that both the basic fold of Thr-102 yeast iso-1-cytochrome c and the region around the site of the mutation are the same as those found in the latter two proteins. It is concluded that the results from structure function studies using the Thr-102 mutant will be applicable to eukaryotic cytochrome c in general. This knowledge allows us to proceed to a description of some mutants of yeast iso-1-cytochrome c in the next paper.

Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of alpha-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolyte-mediated stabilization of the alpha-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the alpha-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the alpha-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.

Cassette mutagenesis was used to produce a library of mutations at the interface of the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c. The library is random and comprises > 98% mutations. Over 11,000 candidates were assayed for function by selecting for the ability of yeast, with the mutated gene as their sole cytochrome c source, to grow on nonfermentable carbon sources. We estimate that approximately 0.5% of the 160,000 total amino acid combinations at these four residues result in a functional cytochrome c. Significant correlations are found between the phenotype of yeast harboring the alleles and both the Dayhoff mutation matrix and transfer free energies (cyclohexane-to-water and n-octanol-to-water). Similar correlations are observed with respect to growth rate. Finally, sequences that are consistent with function follow a binary amino acid pattern.

We have examined the F82Y;C102T variant of Saccharomyces cerevisiae iso-1-cytochrome c using high-resolution proton nuclear magnetic resonance spectroscopy, chemical denaturation, and differential scanning calorimetry. Comparison of proton chemical shifts, paramagnetic shifts, and nuclear Overhauser effects indicates structural changes are localized to the vicinity of position 82. One alteration involves the rearrangement of the side chain of leucine-85. Using many more proton assignments than were available in the initial report [G. J. Pielak, R. A. Atkinson, J. Boyd, and R. J. P. Williams, Eur. J. Biochem. 177, 179-185 (1988)], a second alteration involving an interaction between arginine-13 and tyrosine-82 is observed. The interaction appears to involve a hydrogen bond with the eta-protons of arginine's guanido group acting as donor and tyrosine's phenolic eta-oxygen as acceptor. In spite of this potentially-stabilizing interaction, the free energy of denaturation decreases by approximately 2.4 kcal mol-1. Results are discussed with respect to alterations in the native and denatured states.

Buried within cytochrome c lies a highly-conserved helix-helix interface formed by the perpendicular packing of the C-terminal helix against the N-terminal helix. This interface involves a peg-in-hole interaction between Gly-6 and Leu-94 and an aromatic-aromatic interaction between Phe-10 and Tyr-97. To gain insight into protein design, we investigated the relationship between the sequence of the interface and the physiological function of yeast iso-1-cytochrome c. A library of mutants at positions 94 and 97 of the C-terminal helix was created to examine the effect of novel amino acid combinations. We isolated 45 of the 400 possible amino acid combinations, 32 of which result in a functional cytochrome c. Contrary to evolutionary conservation of the peg-in-hole and aromatic-aromatic interactions, we find that side-chain volume and conservation of aromatic residues do not play an essential role in determining function. Additionally, we find negatively-charged residues within the interface that result in a functional cytochrome c. Examination of the 45 missense mutants indicates that approximately 120 unique combinations are compatible with function. These results show that the interface is flexible. However, truncation of the C-terminal helix at position 94 abolishes function, suggesting that the interface is essential. The correlation observed between our library of mutants and the mutation matrix compiled by Gonnet et al. [Gonnet, G. H., Cohen, M. A., & Benner, S. A. (1992) Science 256, 1443-1445] demonstrates the potential use of the matrix to predict the effect of sequence changes on natural proteins and to optimize the design of novel proteins.

The Ser82 and Phe82 variants of yeast iso-1 cytochrome c were studied by resonance Raman spectroscopy. In both oxidation states, distinct spectral changes were observed for some of those bands in the low-frequency region, which sensitively respond to conformational perturbations of the protein environment of the heme. These bands can be assigned to modes which include strong contributions of vibrations largely localized in the propionate-carrying pyrrole rings A and D. This indicates structural differences in the deeper part of the heme crevice, remote from the mutation site. This conclusion is in line with previous results from X-ray crystallography and NMR spectroscopy. No differences in the resonance-Raman spectra were observed which can be directly correlated with conformational changes of the heme pocket in the vicinity of the mutation site. Temperature-dependent resonance Raman experiments of the oxidized mutants revealed spectral changes which are closely related to those observed for cytochrome c upon adsorption to charged silver surfaces by surface-enhanced resonance Raman spectroscopy. These spectral changes can be attributed to an opening of the heme crevice accompanied by a weakening of the iron-methionine ligand bond. The temperature-dependent conformational transition occurs at approximately 30 degrees C for the Ser82 variant and at about 45 degrees C for the Phe82 variant, implying that the Phe----Ser substitution significantly lowers the thermal stability of the heme pocket. The reduced forms of both mutants are stable up to 65 degrees C.

The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins.

Dipolar paramagnetic shifts for protons of yeast iso-1-cytochrome c have been calculated by using an optimized g-tensor and the X-ray crystallographic coordinates of the reduced form of yeast iso-1-cytochrome c [Louie, G. V., & Brayer, G. D. (1990) J. Mol. Biol. 214, 527-555]. The calculated values are compared with the observed paramagnetic shift determined from over 450 nonequivalent protons that have been assigned in both oxidation states [Gao, Y., Boyd, J., Williams, R. J. P., & Pielak, G. J. (1990) Biochemistry 29, 6994-7003]. There is good agreement between the calculated and the experimental data with a few exceptions. This indicates that, overall, the solution structures must be very similar in both the reduced and oxidized states in solution as is the case in crystals. The differences between observed and calculated shift values for the molecule in solution are most readily explained by slight movement of the heme and certain changes in diamagnetic shift due to small rearrangements of a few residues and some considerable changes in a few hydrogen bonds. It is also known that small differences exist between the structures of the two oxidation states in crystals but the hydrogen-bond changes are not so easily observed there. Structural changes from nuclear magnetic resonance data are in reasonable agreement with those deduced from crystallography, but additional information is clearly available concerning changes in hydrogen bonding.

Oxidative stress and aggregation of the protein alpha-synuclein are thought to be key factors in Parkinson's disease. Previous work shows that cytochrome c with H(2)O(2) causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including alpha-synuclein. Here, we examine the role of each of alpha-synuclein's four tyrosine residues and how the protein's conformation affects covalent oxidative aggregation. When alpha-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild-type-like covalent aggregation in the presence of a denaturant or when alpha-synuclein is present in noncovalent fibrils. We also show that preformed oxidative aggregates are not incorporated into noncovalent fibrils. These data provide insight into how dityrosine may be formed in Lewy bodies seen in Parkinson's disease.

An efficient system for producing human cytochrome c variants is important to help us understand the roles of this protein in biological processes relevant to human diseases including apoptosis and oxidative stress. Here, we describe an Escherichia coli expression system for producing recombinant human cytochrome c. We also characterize the structure, stability, and function of the protein and show its utility for studying apoptosis. Yields of greater than 8 mg of pure protein per liter culture were attained. Circular dichroism spectropolarimetry studies show that the secondary and tertiary structures of the human protein are nearly identical to those of the horse protein, but the human protein is more stable than other eukaryotic cytochromes c. Furthermore, recombinant human cytochrome c is capable of inducing caspase-3 activity in a cell-free caspase activation assay. We use data from this assay along with data from the literature to define the apaf-1 binding site on human cytochrome c.

A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc 1 to cytochrome c (Cc). The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru z -39-Cc. The Ru z -39-Cc derivative has the same steady-state activity with yeast cytochrome bc 1 as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction. Laser excitation of reduced Ru z -39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10 6 s - 1 . The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer. The yield of photooxidized heme c is 20% in a single flash. Flash photolysis of a 1: 1 complex between reduced yeast cytochrome bc 1 and Ru z -39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome c 1 to heme c with a rate constant of 1.4 x 10 4 s - 1 . As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc 1 . The interaction of yeast Ru-39-Cc with yeast cytochrome bc 1 is stronger than that of horse Ru-39-Cc with bovine cytochrome bc 1 , suggesting that nonpolar interactions are stronger in the yeast system.

Proteins are designed to function under crowded conditions where the solute concentration can reach 400 g/L, but they are almost always studied in dilute solutions. To address this discrepancy, we have undertaken a series of studies to determine the effects of high solute concentrations on the thermodynamics of protein equilibria. Recently, we used isothermal titration calorimetry (ITC) to show that high concentrations of mono-, di-, and tetrasaccharides have a small stabilizing effect on the crystallographically defined cytochrome c binding site on yeast ferricytochrome c peroxidase [Morar, A. S., Wang, X., and Pielak, G. J. (2001) Biochemistry 40, 281-285]. Here, we use this technique to show that trisaccharides increase the apparent thermodynamic binding constants for both cytochrome c binding sites on the peroxidase. Mutagenesis studies confirm that the second site includes Asp 148 on the peroxidase. Binding of both cytochrome c molecules is exothermic. The data are interpreted by assuming either the presence or absence of intersite interactions.

We have expressed horse cytochrome c in Escherichia coli. The gene was designed with E. coli codon bias and assembled by using a recursive polymerase chain reaction method. The far-ultraviolet and near-ultraviolet/Soret circular dichroism (CD) spectra show that the structure of recombinant horse cytochrome c is the same as that of the authentic protein. CD-detected thermal denaturation studies were used to measure the thermodynamic parameters associated with two-state denaturation. The free energy of denaturation for the recombinant protein is 10.0 +/- 2.3 kcal mol(-1) at pH 4.6 and 25 degrees C, which agrees with the value for the authentic protein. The expression system will help advance our understanding of the roles of cytochrome c in electron transfer, oxidative stress, and apoptosis by allowing the production of protein variants.

We present a simple and inexpensive method for producing 15N-labeled Saccharomyces cerevisiae iso-1-cytochrome c in Escherichia coli. The labeled protein gives excellent NMR spectra.

Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.

A new ruthenium photoreduction technique was used to measure the formation and dissociation rate constants kf and kd of the high-affinity complex between yeast iso-1-cytochrome c (yCc) and cytochrome c peroxidase compound I (CMPI) over a wide range of ionic strength. These studies utilized Ru-39-Cc, which contains trisbipyridylruthenium attached to the cysteine residue in the H39C, C102T variant of yCc, and has the same reactivity with CMPI as native yCc. kd and kf were measured by photoreducing a small concentration of Ru-39-Cc in the presence of the oxidized yCcIII: CMPI complex, which must dissociate before Ru-39-CcII can bind to CMPI and reduce the radical action. The value of kd for the 1:1 high-affinity complex is very small at low ionic strength,5 s-1 but is increased significantly by binding yCc to a second low-affinity site. However, the low-affinity yCc binding site is not active in direct electron transfer to either the radical cation or the oxyferryl heme in CMPI, and is too weak to play a role in the kinetics at ionic strengths above 70 mM. The value of kd increases to 4000 s-1 at 150 mM ionic strength, while kf decreases from3 x 10(9) M-1 s-1 at low ionic strength to 1.3 x 10(9) M-1 s-1 at 150 mM ionic strength. These studies indicate that the rate-limiting step in enzyme turnover is product dissociation below 150 mM ionic strength and intracomplex electron transfer to the oxyferryl heme at higher ionic strength. The interaction between yCc and CcP is optimized at physiological ionic strength to provide the largest possible complex formation rate constant kf without allowing product dissociation to be rate-limiting. The effects of surface mutations on the kinetics provided evidence that the high-affinity binding site used for the reaction in solution is similar to the one identified in the yCc:CcP crystal structure.

Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S.,Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H.,Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.

A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His39 on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4' methylbipyridine) (bis-bipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 +/- 2.0) x 10(5) s-1, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s-1. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s-1. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 +/- 0.4) x 10(7) M-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.

By combining measurements of the free energy of denaturation of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c with determination of the formal potentials for the native and chemically-denatured states we have determined the free energy of denaturation of the ferro form of the protein. We report that the simplest of all chemical modifications, addition of an electron, increases the stability of ferricytochrome c by approximately 10 kcal mol −1 at 300 K, pH 4.6. This makes reduced cytochrome c one of the most stable proteins yet investigated.

We tested the idea that the aromatic ring on the invariant residue Phe-82 in cytochrome c acts as an electron-transfer bridge between cytochrome c and cytochrome b5. Ru-65-cyt b5 was prepared by labeling the single sulfhydryl group on T65C cytochrome b5 with [4-(bromomethyl)-4'-methylbipyridine][bis(bipyridine)]ruthenium 2+ as previously described [Willie, A., Stayton, P.S., Sligar, S.G., Durham, B., & Millett, F. (1992) Biochemistry 31, 7237-7242]. Laser excitation of the complex formed between Ru-65-cyt b5 and Saccharomyces cerevisiae iso-1-cytochrome c at low ionic strength results in rapid electron transfer from the excited-state Ru(II*) to the heme group of Ru-65-cyt b5 followed by biphasic electron transfer to the heme group of cytochrome c with rate constants of (1.0 +/- 0.2) x 10(5) s-1 and (2.0 +/- 0.04) x 10(4) s-1. Variants of iso-1-cytochrome c substituted at Phe-82 with Tyr, Gly, Leu, and Ile have fast-phase rate constants of 0.4, 1.9, 2.1, and 2.0 x 10(5) s-1 and slow-phase rate constants of 5.3, 3.5, 2.4, and 2.0 x 10(3) s-1, respectively. Increasing the ionic strength to 50 mM results in single-phase intracomplex electron transfer with rate constants of 3.8, 3.1, 3.0, 5.0, and 4.5 x 10(4) s-1 for the wild-type, Tyr, Gly, Leu, and Ile variants, respectively. These results demonstrate that an aromatic side chain at residue 82 is not needed for rapid electron transfer with cytochrome b5. Furthermore, two conformational forms of the complex are present at low ionic strength with fast and slow electron-transfer rates.(ABSTRACT TRUNCATED AT 250 WORDS)

We introduced a novel disulfide bond, modeled on that of bullfrog cytochrome c, into yeast iso-1-cytochrome c. The disulfide spontaneously forms upon purification. A variety of techniques were used to examine the denaturation of this variant and several non-cross-linked controls. Denaturation is reversible and, with the exception of the protein in which the two cysteines are blocked, consistent with a two-state process. Comparison of the calorimetric and van't Hoff enthalpy changes indicates that denaturation is two-state at pH 4.6. Calorimetric and fluorescence-monitored guanidine hydrochloride (GdnHCl) denaturation data indicate that the free energy of denaturation for the cross-linked protein (delta Gd at 300 K) is decreased relative to non-cross-linked controls. The dependence of delta Gd on GdnHCl concentration, the GdnHCl concentration that denatures half the protein, as well as the enthalpy, entropy, and heat capacity changes (mGdnHCl, Cm, delta Hd, delta Sd, and delta Cp, respectively), all decrease in magnitude upon introduction of the cross-link. The decrease in delta Hd and delta Sd were confirmed by monitoring absorbance at several wavelengths as a function of temperature. The cross-link also decreases the pH dependence of these observables. Circular dichroism studies indicate the denatured state of the cross-linked protein possesses more structure than non-cross-linked proteins, and this structure is refractory to increases in temperature and chemical denaturant. We conclude that the diminished values of delta Gd, delta Hd, delta Sd, delta Cp, and mGdnHCl result from the denatured state of the cross-linked variant being more compact and possessing more structure than non-cross-linked controls.

An NMR study of the cytochrome c variant Asn52Ile is used to show how the redox state change in native cytochrome c is coupled to a rearrangement of a proton network which runs through the cytochrome c molecule. The substitution breaks the H-bond network and removes the coupling. The uncovering of this putative proton channel and the connection of changes to it with redox state changes of the iron centre of the protein allows a possible description of the way in which redox energy state changes can be coupled to energization and gating of protons in membranes.

An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1). The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.

Differences in chemical shifts and in nuclear Overhauser effects between the C102T and F82S,C102T variants of Saccharomyces cerevisiae iso-1-cytochrome c in both the reduced and oxidized forms are reported and analyzed. There is evidence for small conformational differences in both oxidation states of the double variant near position 82. Differences in structure are more evident in the oxidized forms of the variants. These differences extend to distant parts of the protein. It is concluded that the oxidized double variant has undergone a small rearrangement of several regions of the protein that are linked by a hydrogen-bond network. It is shown that the rearrangement involves hydrogen bonds associated with the two heme propionates and associated water molecules. The deductions from nuclear magnetic resonance data are compared with the differences in the crystal structures of the reduced forms of wild-type protein and the F82S variant [Louie, G. V., Pielak, G. J., Smith, M., & Brayer, G. D. (1988) Biochemistry 27, 7870-7876].

Resonance assignments for the main-chain, side-chain, exchangeable side chain, and heme protons of the C102T variant of Saccharomyces cerevisiae iso-1-cytochrome c in both oxidation states (with the exception of Gly-83) are reported. (We have also independently assigned horse cytochrome c.) Some additional assignments for the horse protein extend those of Wand and co-workers [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H.,Englander, S. W. (1989) Biochemistry 28, 186-194; Feng, Y., Roder, H., Englander, S. W., Wand, A. J.,Di Stefano, D. L. (1989) Biochemistry 28, 195-203]. Qualitative interpretation of nuclear Overhauser enhancement data allows the secondary structure of these two proteins to be described relative to crystal structures. Comparison of the chemical shift of the backbone protons of the C102T variant and horse protein reveals significant differences resulting from amino acid substitution at positions 56 and 57 and further substitutions between residue 60 and residue 69. Although the overall folding of yeast iso-1-cytochrome c and horse cytochrome c is very similar, there can be large differences in chemical shift for structurally equivalent residues. Chemical shift differences of amide protons (and to a lesser extent alpha protons) represent minute changes in hydrogen bonding. Therefore, great care must be taken in the use of differences in chemical shift as evidence for structural changes even between highly homologous proteins.

In cells, protein-protein interactions occur in an environment that is crowded with other molecules, but in vitro studies are almost exclusively performed in dilute solution. To gain information about the effects of crowding on protein complex formation, we used isothermal titration calorimetry to measure the stoichiometry, the free energy change, and the enthalpy change for the binding of yeast iso-1-ferricytochrome c to yeast ferricytochrome c peroxidase in dilute solution and in solutions crowded with the sugars glucose, sucrose, and stachyose. The stoichiometry is 1:1 under all conditions. The sugars stabilize the complex, but by only 0.1-0.5 kcal.mol(-)(1), and the increased stability is not correlated with the change in enthalpy of complex formation. We then compared the measured stability changes to values obtained from several analyses that are currently used to predict crowding-induced changes in biomolecular equilibria. None of the analyses are completely successful by themselves, and the results suggest that a complete analysis must account for both excluded-volume and chemical interactions.

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.

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.

The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR. The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein. In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved. Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38.The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3198–3197] appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification.

Yeast iso-1-cytochrome c (Cc) mutants have been constructed with Phe, Tyr, Gly, Ser, Leu, and Ile at position 82, each with Thr substituted for Cys at position 102. Their long-range electron transfer with zinc-substituted cytochrome c peroxidase (ZnCcP) has been studied by two kinetic techniques. The charge-separated complex, [(ZnCcP)+,FeIICc] converts to [ZnCcP,FeIIICc] by a single, intracomplex electron transfer step that is not governed by "gating" through possible rapid dissociation of the complex or isomerization (for example, heme-ligand) by FeIICc subsequent to its formation from FeIIICc. In every variant with an aliphatic residue at position 82 of Cc, the rate of this electron transfer process is approximately 10(4) slower at approximately 0 degrees C than for the two variants with aromatic residues.

Phenylalanine 87 of yeast iso-1-cytochromc c (Phe 82 in horse heart and bonito) is phylogenetically conserved1 and occurs near the surface of the protein2. It has been suggested that this residue is directly involved in electron transfer between cytochrome c and cytochrome c peroxidase (CCP)3 and may also control the polarity of the haem environment4. Because Phe residues are not susceptible to chemical modification, no direct means of studying the functional role of Phe 87 has been available, so we have chosen Phe 87 as our initial target here to test the feasibility of using site-directed mutagenesis5 as a means of studying structure–function relationships in cytochrome c. We have changed the codon for Phe 87 to that of either a Ser, a Tyr or a Gly. The mutated genes have been introduced6 into a yeast strain7 lacking both isozymes of cytochrome c. Unlike the recipient strain, transformants grow on a non-fermentable carbon source, indicating that the mutant proteins can reduce cytochrome oxidase7. The purified mutant proteins are similar to wild type with respect to their visible spectra8, 20–70% as active as wild-type protein in the CCP assay9, and their reduction potentials are lowered by as much as 50 mV. Thus Phe 87 is not essential for cytochrome c to transfer electrons but is involved in determining the reduction potential.

Using site-directed mutagenesis, it is possible to prepare many mutants of a protein in a short time, and to uncover differences in function. To understand the changes in function, it is essential to understand the effect(s) of the mutation in terms of structural and dynamic changes. It is particularly important to establish a rapid method for comparing the structure of the mutants with that of the wild-type protein. We propose that a combination of overlayed and difference two-dimensional NOE spectra between the wild-type and mutant protein provide a rapid method for determination of structural similarity. The observation of differences other than those due directly to the field effects of the exchanged side chain allow both local and distant conformational changes to be assessed. Here we compare NOESY spectra from a mutant of yeast iso-1-ferrocytochrome c in which the invariant residue Phe-82 has been changed to a Tyr. We conclude that NMR can show subtle changes in protein structure. Specifically, we show the change must involve the reorientation of the side chain of Leu-85 which is proximal to the mutation. The dynamics of the aromatic side chain at position 82 are shown not to give rise to measurable differences between the wild-type and mutant protein. Structural changes are not propagated to a measurable degree in other parts of the protein.

A prerequisite of electron transfer between two metal centres is effective electronic coupling. For reactions involving metalloproteins the coupling of the redox centres is generally brought about by the formation of a protein-protein complex, the geometric and dynamic properties of which reflect the nature of the protein surface interaction. Two types of intracomplex electron transfer reactivity can be distinguished. In the first type effective electronic coupling and, hence, electron transfer occurs as a direct consequence of protein-protein binding. If any reorganisation in complex structure is required to optimise the coupling it is not rate limiting. It is probable that the reactions of cytochrome c with cytochrome b 5 and cytochrome c with cytochrome c peroxidase fall into this category. The second type is characterised by a relatively large activation barrier to effective redox coupling within the electron transfer complex. In this case protein-protein binding in itself does not give rise to a mutual orientation or combination of protein states which are favourable to electron transfer. The kinetic barrier may be related to unfavourable electronic spin-states or a relatively large redox centre separation in such a way that a change in protein conformation or orientation is required before electron transfer can occur. Intracomplex reactions of this type may be susceptible to catalysis or control by additional molecular species which facilitate the necessary conformational or electronic changes.

Oxidative stress and aggregation of the protein μ-synuclein are thought to be key factors in Parkinson's disease. Previous work shows that cytochrome c plus H2O2 causes tyrosine-dependant in vitro peroxidative aggregation of proteins, including μ-synuclein. Here, we examine the role of each of α-synuclein's four tyrosine residues and how the protein's conformation affects covalent oxidative aggregation. When μ-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild type-like covalent aggregation in the presence of a denaturant or when μ-synuclein is present in non-covalent fibrils. We also show that pre-formed oxidative aggregates are not incorporated into non-covalent fibrils. These data provide insight as to how dityrosine may be formed in Lewy bodies seen in Parkinson's disease.