1H-NMR studies of the coordination geometry at the heme iron and the electronic structure of the heme group in cytochrome c-552 from Euglena gracilis.

The 1H-NMR lines of heme c in reduced and oxidized cytochrome c-552 from Euglena gracilis were individually assigned and the coordination geometry of the axial ligands was investigated. The electronic structure of the heme and the chirality of the axially bound methionine were found to be of the same type as in mammalian cytochrome c, but different from cytochrome c-551 from Pseudomonas aeruginosa. These results provide additional support for a previously proposed correlation between the chirality of attachment of the axial methionine and the electronic wave functions in oxidized cytochromes of the c type. Comparison of mammalian cytochrome c, cytochrome c-551 and cytochrome c-552 indicates that the chirality of the axially bound methionine is not linked with the evolutionary increase of the polypeptide chain length.

1H NMR studies of the heme iron coordination in cytochrome c-552 from Euglena gracilis.

The coordination of the heme iron in cytochrome c-552 from Euglena gracilis was investigated by 1H NMR studies at 360 MHz. The data imply that the axial heme ligands are His-14 and Met-56 in both the oxidized and the reduced protein. Studies of mixed solutions of ferro- and ferricytochrome c-552, which provided much of the information on the heme structure, also showed that the intermolecular electron exchange is characterized by a bimolecular rate constant of 5-10(6) mol-1-s-1 at 29 degrees C, which is three orders of magnitude faster than the corresponding reaction in solutions of mammalian cytochromes c.

The role of histidines 26 and 33 in the structural stabilization of cytochrome c.

Comparative studies of the importance of the two histidines of rat cytochrome c that are not ligands of the heme iron, for the stability of the protein, were carried out by site-directed mutagenesis. Histidine 26 was substituted by valine and the resulting effects on the stability of the Met-80-sulfur to heme iron bond to changes in pH and temperature, and of the global stability of the protein to unfolding in urea solutions, were measured. It is suggested that the loss of the hydrogen bond between the His-26 imidazole and the backbone amide of Asn-31 caused the observed decreases in local stability; and that, in addition, the elimination of the hydrogen bond between this imidazole and the carbonyl of Pro-44 resulted in an increase of the mobility of the lower loop (residues 41-47) on the right side of the protein and of its distance from the middle loop (residues 26-31), probably leading to greater hydration of the interior right side of the molecule. These changes resulted in a decrease in the global stability of the protein. Further mutation of Asn-52 to Ile led to a total recovery of the wild-type stability of the sulfur-iron bond, and a partial restoration of the global stability of the protein. Substitution of Phe for His-33 did not alter the sulfur-iron bond but caused a pronounced increase in the global stability of the protein. It is suggested that this effect results from hydrophobic interaction of the Phe-33 side chain with the lower loop on the right side of the protein. Such an interaction also explains the observation that the same mutation reversed the loss of global stability caused by substitution of Val to His-26, but did not restore the strength of the sulfur-iron bond that this mutation had brought about.

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation.

Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric state, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bond formation, while the TP Lys-79 cytochrome c dimerized with a half-time of approx. 3 h, both recovering the native-like iron ligation. By contrast, in the ferrous state the monomeric state and the native ligation were preserved in all cases, indicating that the affinity of the cytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capability of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase.

The reactivity of cytochrome c with soft ligands.

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron-methionine sulfur bond of this heme protein is enhanced by delocalization of the metal t2g electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.

Myopathy, lactic acidosis, and sideroblastic anemia: a new syndrome.

We describe 2 sibs (brother and sister) with myopathy, sideroblastic anemia, lactic acidosis, mental retardation, microcephaly, high palate, high philtrum, distichiasis, and micrognathia. Very low levels of cytochromes a, b, and c were detected in the patients' muscle mitochondria. Deposition of iron within the mitochondria of bone marrow erythroblasts was observed on electron microscopy. Irregular and enlarged mitochondria with paracrystalline inclusions were also seen on electron microscopy of the patients' muscle specimen. Examination of DNA from the affected sibs showed no deletions in the mitochondrial DNA nor the mutations identified in the syndromes of mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) or myoclonus, and epilepsy associated with rugged-red fibers (MERRF). Since the parents were first cousins and 2 of 6 sibs (male and female) were affected, we suggest that the syndrome expressed by our patients represents a previously unknown autosomal recessive disorder that includes mitochondrial myopathy, lactic acidosis, and sideroblastic anemia.

The source of heterogeneity in the heme vicinity of ferricytochrome c.

Heterogeneity in the heme vicinity of ferricytochrome c was reported to be detectable by a split of the NMR signal of the heme methyl 3 group [P.D. Burns and G.N. La Mar, J. Am. Chem. Soc. 101 (1979) 5844]. Using cytochrome c mutants and computer simulations of the native and mutated cytochromes, the source of this heterogeneity is found to originate from the His-33 residue motions. The H33F mutation abolished the NMR split and computer simulations of the H33F mutant revealed a narrower distribution of fluctuations of the radius of gyration, suggesting a more rigid structure due to the mutation. The stabilization of the mutant was further demonstrated by a reduction in the H33F mutant of 4 Kcal/mol in the calculated interaction energy between residue 33 and the rest of the cytochrome, in keeping with known experimental results.