Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy.

Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.

Still a puzzle: why is haem covalently attached in c-type cytochromes?

c-Type cytochromes are a group of proteins with diverse structures and functions. Their common feature is covalent attachment of haem to one or more CXXCH motifs. There does not seem to be a single advantageous reason for this covalent attachment.

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.

Age-related changes in the fluorescence of melanin and lipofuscin granules of the retinal pigment epithelium: a time-resolved fluorescence spectroscopy study.

The photophysical properties of purified populations of melanin and lipofuscin granules from human retinal pigment epithelium, and their changes with donor age, have been investigated using high-sensitivity time-resolved fluorescence spectroscopy techniques with picosecond gating capabilities. The overall fluorescence intensity of both melanin and lipofuscin granules clearly increased with increasing donor age, the increase being most marked for melanin. In all granule populations the fluorescence decays were multiexponential with subnanosecond and nanosecond decay components. The resultant time-integrated and time-gated spectra also exhibited marked age-variations for each type of granule. Young melanin showed spectral patterns similar to those of bovine melanin, while a yellow-orange fluorescence band appeared in melanin samples from older age groups. Lipofuscin granules exhibited a blue, a yellow and an orange band whose relative amounts were age-related. The results demonstrate the potential of time-resolved techniques for discriminating fluorophores in vitro and in situ, and have confirmed results previously obtained using extraction techniques. Furthermore, the ability of this technique to identify and quantify individual fluorophores within granules may provide an important insight into the origin and development of lipofuscin within the retinal pigment epithelium and ultimately into the mechanisms of age-related retinal diseases.

Interfacial redox processes of cytochrome b562.

The anionic soluble heme protein cytochrome b(562) was electrostatically immobilised on Ag electrodes coated with positively charged self-assembled monolayers of amino-terminated alkanethiols. The structure of the heme pocket, the redox equilibria, and the electron transfer dynamics were studied by stationary and time-resolved surface enhanced resonance Raman spectroscopy, complemented by cyclic voltammetry measurements of the interfacial redox process. The conformational and redox equilibria of the immobilised protein are compared to those of the cationic heme protein cytochrome c immobilised on negatively charged electrode coatings. Similarities and differences can be rationalised in terms of the respective electric fields at the interfaces of amino- and carboxyl-terminated electrode coatings. The heterogeneous electron transfer rate of cytochrome b(562) only slightly increases with decreasing thickness from ca. 20 to 11 A, implying that the electron tunneling is not the rate-limiting step. In contrast to cytochrome c on carboxyl-terminated monolayers, this behaviour cannot be attributed to protein re-orientation gating the heterogeneous electron transfer. Instead, it may reflect the interplay between interprotein electron transfer and heterogeneous electron transfer via protein orientations exhibiting particularly high tunneling probabilities for the electron exchange with the electrode.

Sensitization and multiplicative noise in the descending contralateral movement detector (DCMD) of the locust.

Spike discharges from the descending contralateral movement detector (DCMD) were recorded extracellularly from the ventral nerve cord of the locust in complete darkness, in response to dim flashes of constant-intensity light, and in response to pairs of identical flashes presented different intervals apart. Three phenomena were discovered: novel long-term sensitization which changes the DCMD's sensitivity to light, a multiplicative cascade process driven by shot events, and the suppression of the spike discharge shortly after a dim flash. The DCMD's spike discharge is stochastic. It can be considered as a two-stage cascade process producing intrinsic multiplicative noise. An effective photon, or thermal isomerization in complete darkness, produces an unseen shot event which in turn initiates a random number of DCMD spikes in a cluster. A short initiates a variable number of spikes when it directs the rate of a Poisson process. The results of statistical analyses are consistent with this model when the amplitudes of shot events are variable. The transmission efficiency is low because at least 2.4-9.6 quantum bumps are required to produce one extra DCMD spike. The DCMD has a constant mean discharge rate of 0.25-1.5 spikes/s in complete darkness. Clustering about particular points in time (shots) leads to a lack of independence between interspike intervals, and the overdispersion of interspike interval and number distributions compared with those from a simple Poisson process. The mean cluster size is 1.3-1.6 spikes in darkness. Similar clustering was found in response to flashes of light. A dim flash changes the DCMD's sensitivity to light, even at threshold when no spike discharge results. Sensitization occurs because the average number of shot events produced by isoquantal flashes depends on the history of visual stimulation. This contributes to the nonlinear response-intensity function. The evolution of sensitization is roughly constant in different DCMD cells, lasting approximately 3 s after a flash. Sensitization was observed in response to light only, presumably because the intensity of dark-light is too low. It is proposed that sensitization is associated with a set of processes or molecular state in the presynaptic region of a chemical synapse.

Bis-methionine ligation to heme iron in mutants of cytochrome b562. 2. Characterization by NMR of heme-ligand interactions.

Previous work has shown that, in variants of cytochrome b562 containing the H102M mutation, methionine residues provide both axial ligands to the heme iron. NMR spectroscopic studies of such bis-methionine-coordinated cytochrome have not previously been feasible, since the only other cytochrome with such a ligand arrangement, bacterioferritin, is too large to be studied by current NMR methods. The present work provides the first NMR characterization of 6-coordinate, bis-methionine-ligated heme centers in both ferrous and ferric oxidation states. We have used one and two dimensional, homonuclear NMR spectroscopy to assign the proton resonances of the heme group and ligand side chains in the reduced, cytochrome b562 variants, H102M and covR98C/H102M. The latter protein has heme covalently attached to the protein, and our results prove that the covalent linkage is a c-type thioether bond formed between the cysteine at residue 98 and the heme 2-vinyl group. Spectra of the ferrous H102M variant are consistent with the presence of two species differing in the orientation of the heme in the protein. We have interpreted results from NOESY experiments on the ferrous covR98C/H102M protein in terms of the conformation of the two methionine side chains, and we present a model for the structure of the heme ligand arrangement. The Met7 side chain adopts an extended conformation almost identical to that observed in the wild type protein with R stereochemistry at the chiral sulfur ligand. The Met102 side chain has a different, buckled side chain conformation and has S stereochemistry at the chiral center. Our NMR derived model is consistent with the spectroscopic data presented in the previous paper. Studies on the ferric forms of these proteins confirm that the double variant at low pH has a "stable" bis-methionine ligation arrangement, but that it is a thermal mixture of species with differing spin states. No hyperfine coupled proton resonances can be identified in spectra of the high-spin forms of either of these proteins.

Direct electrochemical probes of redox protein and redox enzyme structure and function.

The direct electrochemistry of redox proteins has been achieved at a variety of electrodes, including modified gold and pyrolytic graphite. Careful design of electrode surfaces and electrolyte conditions are required for the attainment of rapid and reversible protein-electrode interaction. Protein-protein complexes can be studied by using selective electrode surfaces. The "well-behaved" electrochemistry of redox proteins can be exploited by coupling the electrode reaction to enzymes for which the redox proteins act as cofactors. The electron-transfer reactions of more complex systems, such as redox enzymes, are now being examined.

Coupled oxidation of heme covalently attached to cytochrome b562 yields a novel biliprotein.

A variant of Escherichia coli cytochrome b(562) with covalently attached heme can be converted to a biliverdin-containing protein in two distinct stages by coupled oxidation and acid hydrolysis. The first stage of coupled oxidation yields a stable verdoheme-containing protein. This verdoheme protein is unusual in three respects. First, the verdoheme group is covalently bound to the protein through a c-type thioether linkage. Second, the oxidation stops at the verdoheme stage, and finally, this is the first report of verdoheme generated from a heme protein with exclusive methionine ligation to the heme iron. In addition, the oxidation process does not require denaturation of the protein. The product has been characterized by optical spectroscopy, ESI mass spectrometry, and (1)H NMR. The NMR data show that the predominant product is the result of oxidation at the alpha-meso carbon. A collective evaluation of data on the topic suggests that the electronic structure of the heme, not protein steric effects, is the main factor in controlling the regiospecificity of the oxidation site. In the second stage of conversion to a biliprotein, we demonstrate that the verdoheme ring can be opened by treatment with aqueous formic acid to give alpha-biliverdin covalently attached to the folded protein. This product, a protein-bound linear tetrapyrrole as characterized by optical spectroscopy and mass spectrometry, is an example of a phycobilin chromophore that has not been observed previously.

Proton titration curve of yeast iso-1-ferricytochrome c. Electrostatic and conformational effects of point mutations.

The proton titration curves of yeast iso-1-ferricytochrome c and selected point mutants of this protein have been determined between pH 3 and 11 at 10 and 25 degrees C with a computer-controlled titration system. Initial titration of the wild-type protein to acidic pH followed by subsequent titrations to alkaline and then acidic pH demonstrates hysteresis, with one more group (28.7) titrating between pH 11 and 3 than originally titrated (27.7) between pH 3 and 11. Initial titration to alkaline pH, however, resulted in observation of the same number of groups in both directions of titration (28.7 vs 28.6). At 10 degrees C, 7.5 fewer groups were found to titrate over the same range of pH. Titration curves obtained for six cytochrome c mutants modified at Arg-38, Phe-82, Tyr-48, and Tyr-67 were analyzed by subtraction of the corresponding titration curve for the wild-type protein to produce difference titration curves. In most cases, the effects of these mutations as revealed in the difference titration curves could be accounted for as either the result of introduction of an additional group titrating within this pH range, the result of a change in the pK of a titrating residue, and/or the result of a change in the pK for either the first acidic or the first alkaline protein conformational transition. In addition to demonstration of the electrostatic consequences of the mutations in cytochrome c studied here, this study establishes the general usefulness of precise proton titration curve analysis in the characterization of variant proteins produced through recombinant genetic techniques.

Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions.

Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5. With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH. The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins. Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small. Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation. Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different. In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied. Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)

Conversion of cytochrome b562 to c-type cytochromes.

Cytochrome b562 from the periplasm of Escherichia coli is the only member of a family of cytochromes sharing the 4-alpha-helical bundle structural motif that does not have a covalently bound heme. We have introduced cysteine residues into the amino acid sequence of cytochrome b562 in positions homologous to those found in the other members of the family, generating the ubiquitous heme-binding peptide (-C-X-Y-C-H-) found in virtually all c-type cytochromes. The resulting single-cysteine-containing mutants, R98C and Y101C, together with the double mutant combining both of these mutations have been expressed into the periplasm of E. coli. The apo- and holoprotein products of each mutation have been isolated, and all the mutants produce multiple species with covalently attached heme. Results from ion exchange chromatograph, optical spectroscopy, SDS gel electrophoresis, and electrospray mass spectrometry identified those species that appear to be cytochrome b562 holoprotein with thioether covalent linkages to the heme as the only difference in chemical composition between them and the wild-type protein. Results from 1H-NMR experiments prove the existence of the expected c-type covalent bonds in each of these proteins and show that the structure of the heme pocket is not significantly perturbed by the covalent modification(s). These proteins all have perturbed optical spectra, compared with those of the wild-type protein, that are consistent with the modifications but are still characteristic of six-coordinate, low-spin cytochromes with Met-His ligation to the heme iron in both oxidation states.

Direct electrochemistry of protein-protein complexes involving cytochrome c, cytochrome b5, and plastocyanin.

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.

Electron transfer reactions of metalloproteins at peptide-modified gold electrodes.

The electron transfer reactions of four small redox proteins, cytochrome c. ferredoxin, plastocyanin and azurin, have been investigated at novel peptide-modified gold electrodes. These proved to be effective and selective in facilitating electron transfer. Good, quasi-reversible electron transfer was achieved selectively at different peptide-protein configurations by changing the pH or the ionic strength of the solution. The use of peptides as promoters for protein electrochemistry opens up the possibility of designing very specific electrode surfaces for larger molecules like enzymes.

(15)N backbone dynamics of ferricytochrome b(562): comparison with the reduced protein and the R98C variant.

The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy. The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed. (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant. In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Biochemistry 39, 1499-1514]. The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species. The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2. Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein. The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562). In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing. The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity.

Structural consequences of b- to c-type heme conversion in oxidized Escherichia coli cytochrome b562.

An NMR characterization of the 98Arg --> Cys variant of iron (III)-containing cytochrome b562 from Escherichia coli has been performed and the solution structure obtained. This variant has a covalent bond between the heme and Cys 98, thus mimicking the heme binding in cytochrome c. The R98C cytochrome is shown to have a significantly increased stability, compared to that of wild type, toward thermal and chemical denaturation. In water at 20 degrees C it is 5.60 kJ mol-1 more stable than the WT protein, measured by equilibrium guanidine hydrochloride denaturation. The structure has been obtained through two-dimensional total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments and through three-dimensional NOESY-15N heteronuclear multiple quantum coherence (HMQC). By these methods, 85% of protons and 100% of backbone nitrogens were assigned. 2145 meaningful nuclear Overhauser effects (NOEs) (20 NOEs per residue), 45 backbone 3J values, and 397 pseudocontact shifts were used to obtain a family of 35 members, which were then energy-minimized. The root-mean-square deviation (RMSD) with respect to the average structure is 0.50 +/- 0.07 for the backbone and 1.01 +/- 0.08 for the heavy atoms. The magnetic anisotropy resulting from analysis of the pseudocontact shifts indicates an anisotropy that is an intermediate between that of the wild-type, which is the smallest, and cytochrome c. The g values confirm a higher anisotropy of the variant with respect to the wild-type protein. The chirality of the heme 2 alpha carbon is the same as that in all naturally occurring cytochromes c. The overall secondary structure and tertiary structure are very similar to the wild type. The removal of Arg 98 causes a change in the pH-dependent properties. The pKa, proposed to be due to deprotonation of the coordinated histidine, is 1.5 units higher than in the wild type, consistent with the lack of the positive charge of Arg 98 close to the ionizable group. This is further support for the coordinated histidine being the titratable group with an alkaline pKa in the wild-type protein. The pattern of the shifts of the heme methyl groups is different than in the wild-type protein, presumably due to alteration of the electronic structure by the presence of the covalent bond between the protein and the heme. The difference in stability between the variant and wild-type protein is discussed in terms of the structural information.

The solution structure of oxidized Escherichia coli cytochrome b562.

The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pKa values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity.

Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.

Bis-methionine ligation to heme iron in mutants of cytochrome b562. 1. Spectroscopic and electrochemical characterization of the electronic properties.

We have generated mutants of cytochrome b562 in which the histidine ligand to the heme iron (His102) has been replaced by a methionine. The resulting proteins can have bis-methionine coordination to the heme iron, but the stability of this arrangement is dependent on oxidation state and solution pH. We have used optical, MCD, and EPR spectroscopies to study the nature of the heme coordination environment under a variety of conditions. Optical spectra of the reduced state of the single variant, H102M, are consistent with bis-methionine ligation. In its oxidized state, this protein is high-spin under all conditions studied, and the spectroscopic properties are consistent with only one of the methionine ligands being coordinated. We cannot identify what, if anything, provides the other axial ligand. A double variant, R98C/H102M (in which the heme is covalently attached to the protein through a c-type thioether linkage), is also bis-methionine coordinated in the ferrous state, but has significantly different properties in the oxidized state. With a pKa of 7.1 at 20 degrees C, the protein converts from a low-spin, 6-coordinate heme protein at low pH, to a high-spin species, similar to the high-spin species observed for the single variant. Our spectroscopic data prove that the low-spin species is bis-methionine coordinated. The reduction potential of this bis-methionine species has been measured using direct electrochemical techniques and is +440 mV at pH 4.8. The electrochemistry of these proteins is complicated by coupled coordination-state changes. Proof that the ferrous state is bis-methionine coordinated is provided by NMR results presented in the following paper.

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)