The alkaline conformation (state IV) of yeast iso-1-ferricytochrome c and variants in which selected lysyl residues were replaced with alanyl residues has been studied by 1H NMR spectroscopy, electronic spectroscopy, EPR spectroscopy, direct electrochemistry, pH-jump kinetics, and temperature-dependent circular dichroism spectroscopy. On the basis of the NMR studies, Lys73 and Lys79 are shown to replace Met80 as the axial ligand in the two conformers of state IV that were detected in previous studies (Hong, X. L.; Dixon, D. W. FEBS Lett. 1989, 246, 105−108; Ferrer, J. C.; Guillemette, J. G.; Bogumil, R.; Inglis, S. C.; Smith, M.; Mauk, A. G. J. Am. Chem. Soc. 1993, 115, 7507−7508). The pK a for the conformational equilibrium between state III (native conformation) and state IV of the wild-type protein (8.70(2)) is found to be intermediate between that of the Lys73 bound conformer (8.44(1)) and that of the Lys79 bound conformer (8.82(2)) (0.1 M NaCl, 25 °C) as are the kinetic parameters for the conversion of native protein to each of the two alkaline conformers and the midpoint reduction potentials of the two alkaline forms. The EPR spectra of the Lys73Ala and Lys79Ala variants permit interpretation of the corresponding spectrum of the wild-type protein as the sum of two component conformers. The Lys79Ala variant is slightly more susceptible to thermal denaturation at pH 6.15, but the Lys73Ala variant is less thermally stable than the wild-type cytochrome or the Lys79Ala variant at alkaline pH. The Lys73Ala/Lys79Ala double variant retains the spectroscopic characteristics of the native cytochrome at moderately high pH and appears to undergo a change of axial ligation only under more alkaline conditions (pK a ∼ 10.5). This observation suggests that the coordination of the amine ligands is a significant contribution toward the driving force for formation of the state IV conformers. These results establish the axial ligation of yeast iso-1-ferricytochrome c state IV, characterize the kinetics with which state III converts to state IV, and establish the electrochemical properties and thermal stabilities of the two conformers that constitute state IV. The results of this work are discussed with reference to pH-dependent structural behavior of other proteins, the mechanism by which these conformers of the ferricytochrome are formed, and the relationship of the present results to those reported previously for the formation of state IV from state III.

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.

Residue Asn57 of bovine liver cytochrome b5 has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C. A combination of electronic spectroscopy, 'H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives. The red form A (Em = -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57 and the a carbon of the heme 4-vinyl group. Form B, has a covalently bound heme group coupled through a thioether linkage involving the 13 carbon of the heme 4-vinyl group. Form BI, is similar to B, except that the sulfur involved in the thioether linkage is oxidized to a sulfoxide. The green form C (Em = 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin. A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed.While iron-protoporphyrin IX is ubiquitous as a noncovalently bound prosthetic group in a wide variety of electrontransfer proteins and enzymes, other metalloporphyrins that vary in the type and degree of substitution in the macrocycle are known to be important in biological systems (1). These include hemes a, c, o, and d (2), the last of which represents the general class of metallochlorins-i.e., metalloporphyrins in which one pyrrole ring is reduced.Site-directed mutagenesis is an established method for generation of metalloprotein variants as a means of gaining mechanistic insight into electron-transfer processes (3). We now report a cytochrome b5 variant that is representative of a class of heme protein mutants in which the native prosthetic group undergoes specific chemical modifications. As these modifications are related to the classical designations ofheme proteins, we refer to this phenomenon in which transformation between heme types results from site-specific protein mutation as transmutation. Characterization of the present cytochrome b5 variant and its properties, combined with similar studies of subsequent variants based on related principles, promises to provide useful insight into the mechanisms involved in the covalent attachment of the heme prosthetic group to cytochromes c and in chlorin biosynthesis. The principles to be learned from this variant should lead to the design of metalloproteins with specifically modified prosthetic groups possessing useful enzymatic and/or electron-transfer properties.

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The electrostatic character of cytochrome c-cytochrome c peroxidase complex formation has been studied by potentiometric titration between pH 5.5 and 7.75. Potentiometric data obtained at ionic strength > or = 100 mM were adequately analyzed in terms of 1:1 complex formation while the simplest model capable of fitting similar data obtained at lower ionic strength involves the assumption of two inequivalent binding sites for the cytochrome on the peroxidase. The stability of cytochrome c binding at the high-affinity site is ca. three orders of magnitude greater than that observed for the low-affinity site and is optimal between pH 6.75 and 7. The electrostatic properties of the two binding sites are distinctly different because, at most values of pH, binding of cytochrome c to the high-affinity site results in proton release while binding of the cytochrome to the low-affinity site results in proton uptake. Furthermore, binding of the cytochrome to the low-affinity site appears to be least stable in the pH range where binding to the high-affinity site is optimal. Interestingly, the binding parameters derived from these measurements were independent of temperature, consistent with a substantial entropic contribution to complex stability. Ferricytochrome c binds to the peroxidase with a slightly greater affinity than does ferrocytochrome c, and no evidence for specific anion effects on complex stability was observed. At low ionic strength (< or = 50 mM) and high pH (7.75), the interaction of the two proteins is more complex and cannot be adequately analyzed in terms of the two-site model.