Cyt c (cytochrome c) has been traditionally envisioned as rapidly diffusing in two dimensions at the surface of the mitochondrial inner membrane when not engaged in redox reactions with physiological partners. However, the discovery of the extended lipid anchorage (insertion of an acyl chain of a bilayer phospholipid into the protein interior) suggests that this may not be exclusively the case. The physical and structural factors underlying the conformational changes that occur upon interaction of ferrous cyt c with phospholipid membrane models have been investigated by monitoring the extent of the spin state change that result from this interaction. Once transiently linked by electrostatic forces between basic side chains and phosphate groups, the acyl chain entry may occur between two parallel hydrophobic polypeptide stretches that are surrounded by positively charged residues. Alteration of these charges, as in the case of non-trimethylated (TML72K) yeast cyt c and Arg91Nle horse cyt c (where Nle is norleucine), led to a decline in the binding affinity for the phospholipid liposomes. The electrostatic association was sensitive to ionic strength, polyanions and pH, whereas the hydrophobic interactions were enhanced by conformational changes that contributed to the loosening of the tertiary structure of cyt c. In addition to proposing a mechanistic model for the extended lipid anchorage of cyt c, we consider what, if any, might be the physiological relevance of the phenomenon.

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We have measured the electronic circular dichroism (ECD) of the ferri- and ferro-states of several natural cytochrome c derivatives (horse heart, chicken, bovine, and yeast) and the Y67F mutant of yeast in the region between 300 and 750 nm. Thus, we recorded the ECD of the B- and Q-band region as well as the charge-transfer band at approximately 695 nm. The B-band region of the ferri-state displays a nearly symmetric couplet at the B0-position that overlaps with a couplet 790 cm-1 higher in energy, which we assigned to a vibronic side-band transition. For the ferro-state, the couplet is greatly reduced, but still detectable. The B-band region is dominated by a positive Cotton effect at energies lower than B0 that is attributed to a magnetically allowed iron-->heme charge-transfer transition as earlier observed for nitrosyl myoglobin and hemoglobin. The Q-band region of the ferri-state is poorly resolved, but displays a pronounced positive signal at higher wavenumbers. This must result from a magnetically allowed transition, possibly from the methionine ligand to the dxy-hole of Fe3+. For the ferro-state, the spectra resolve the vibronic structure of the Qv-band. A more detailed spectral analysis reveals that the positively biased spectrum can be understood as a superposition of asymmetric couplets of split Q0 and Qv-states. Substantial qualitative and quantitative differences between the respective B-state and Q-state ECD spectra of yeast and horse heart cytochrome c can clearly be attributed to the reduced band splitting in the former, which results from a less heterogeneous internal electric field. Finally, we investigated the charge-transfer band at 695 nm in the ferri-state spectrum and found that it is composed of at least three bands, which are assignable to different taxonomic substates. The respective subbands differ somewhat with respect to their Kuhn anisotropy ratio and their intensity ratios are different for horse and yeast cytochrome c. Our data therefore suggests different substate populations for these proteins, which is most likely assignable to a structural heterogeneity of the distal Fe-M80 coordination of the heme chromophore.

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Cytochrome c binds ATP with marked specificity at a site that contains the evolutionarily invariant residue Arg-91. The binding of ATP to this site was studied using equilibrium gel filtration, equilibrium dialysis and affinity chromatography. At physiological ionic strength the affinity is such that the major change in occupancy coincides with the normal cellular ATP concentration range, and the degree of saturation is proportional to the ratio of [ATP]/[ADP]. The specificity of binding at this site is more a function of the degree of phosphorylation of the nucleotide, than of the nature of the nucleoside moiety. Thus under physiological conditions the degree of occupancy of this site is proportional to the energy state of the cell, providing a means for the regulation of the respiratory chain which is sensitive to cytoplasmic ATP levels.

The effect of the protein matrix on the standard potential of a buried redox center has been investigated by using a selection of mutants and chemical derivatives in Saccharomyces cerevisiae cytochrome c isoform 1. Assuming only local structural perturbation and no alteration of the iron-ligation chemistry, ⌬E m 0 can be regarded as a measure of the difference in polypeptide solvation of the heme charge, which reflects the dielectric properties of the protein. In double mutants Y67F/N52I Y67F/ N52V, where most of the hydrogen bond network in the heme crevice is eliminated, ⌬S redox compares to the wild type. This indicates that a fully consistent hydrogen bond network has a similar polarizability as an apolar matrix. We therefore argue that the variability in net dielectric susceptibility arises from conformational polarizability, a factor that is not a function of atomic properties and coordinates and is therefore hard to predict using conventional physical relationships.There is still much to learn about the factors determining the E m 0 Ј of redox-active proteins. Determinants of E m 0 Ј such as local structural effects (1) as well as the electrostatic landscape of the polypeptide (2) (including the protein's own charges (3, 4), dipolar matrix (5, 6), and surrounding solvent molecules (7)) have already been identified. However, implementation of this knowledge into models does not reliably reproduce experimental observations.The standard redox potential (E m 0 Ј) reflects the thermodynamics of the equilibrium between redox states. The energetics of rearrangement between redox states will therefore reflect the ability of a system to polarize in an electrostatic field. Assuming that the nature of the iron-ligand interaction remains unchanged, the values of ⌬E m 0 Ј of mutation can be regarded as a measure of change in the polarizability, or dielectric response, to the charge of the redox center.With virtually no large redox-dependent conformational change or ligand rearrangement (8 -12), cytochrome c is a suitable model to investigate the contribution of individual residues to the dielectric properties of the protein. Using three positively charged carboxyamidomethyl-methionine sulfonium ion (CAMMS) 1 derivatives 2 (Fig. 1A) generated from a mutational methionine scan (13), a series of buried mutations at position 52 (Fig. 1B) (14) and the point mutations Y67F, Y67F/ N52I, and Y67F/N52V (15), an attempt to rationalize ⌬E m 0 Ј mut in terms of polarizability of the protein matrix is made. EXPERIMENTAL PROCEDURESDetermination of Redox Potential-The measurement of redox potential was made using the method of mixtures (16) in 50 mM potassium phosphate (pH 7.0). The redox state of the sample protein was assayed in a range of redox buffers set by the ratio of ferro/ferricyanide in solution using an E m 0 Ј for the couple of ϩ0.43 V. Thermodynamics of Oxidoreduction-Temperature dependence of the equilibrium between the reduced and oxidized form of the cytochrome c was measured in 50 mM potassium phosphate (pH 7.0) wit...

Eukaryotic cytochrome c possesses an ATP-binding site of substantial specificity and high affinity that is conserved between highly divergent species and which includes the invariant residue arginine''. Such evolutionary conservatism strongly suggests a physiological role for ATP binding that demands further investigation. We report the preparation of adducts of the protein and the affinity labels 8-azido adenosine 5'-triphosphate, adenosine S-triphosphate-T,3'-diaIdehyde, and 5'-p-fluorosulfonylbenzoyladenosine. The two former reagents were seen to react at the arginine9'-containing site, yet the reaction of the latter, although specific, occurred elsewhere, suggesting caution is necessary in its use. None of the adducts displayed significant modification of global structure, stability, or physicochemical properties, leading us to believe that the 8-N3-ATP and oATP adducts are good stabilized models of the noncovalent interaction; yet modification led to significant, and sometimes pronounced, effects on biological activity. We therefore propose that the role of ATP binding to this site, which we have shown to occur when the phosphorylation potential of the system is high under the equivalent of physiological conditions, is to cause a decrease in electron flow through the mitochondrial electron transport chain. Differences in the degree of inhibition produced by differences in adduct chemistry suggest that this putative regulatory role is mediated primarily by electrostatic effects.