Heme Crevice Research Articles

NMR study of haem exchange reaction of native myoglobin and haemoglobin

AbstractA proton NMR study of the haem exchange reactions of native myoglobin and haemoglobin with the various haems revealed that the haem displacement reaction occurs in native haemoproteins as has been detected in the reconstituted haemoproteins. In the presence of an excess amount of the modified haem, protohaem was excluded from a native haemoprotein to yield a reconstituted haemoprotein containing the modified haem, suggesting that the conformational fluctuations in native haemoprotein are large enough to dissociate its haem from the haem crevice. Although the haemoglobin subunits and myoglobin exhibit very similar three‐dimensional structures as revealed by x‐ray diffraction studies, the rates of the haem exchange reactions in myoglobin were larger than those in the haemoglobin β‐subunit. Such differences in the dynamic properties suggest that these haemoproteins have different interactions between the haem and amino acid residues inside the haem pocket.

Mutagenesis of the conserved lysine 14 of cytochrome c-550 from Thiobacillus versutus affects the protein structure and the electron self-exchange rate.

The lysine residue K14 of cytochrome c-550 of Thiobacillus versutus has been mutated to a glutamine (Q) and a glutamate (E) residue. These mutations have a minimal effect on the pKa for replacement of the methionine ligand (the "alkaline transition"), indicating that a presumptive salt bridge between K14 and E11 does not help stabilize the native form. This is in contrast with mitochondrial cytochrome c, where the homologous K13 forms a structurally important salt bridge with glutamate 90. The NMR signals of protons close to the heme iron in wild-type and mutant ferricytochrome c-550 shift considerably with increasing ionic strength. These effects resemble those seen in mitochondrial cytochrome c upon addition of salt and upon complex formation with redox partners. It is likely that electrostatic screening of positive charges near the heme crevice leads to a slight redistribution of the electron density in the heme. At low ionic strength the NMR spectrum of wild-type cytochrome c-550 shows broad peaks. Line widths decrease upon addition of salt up to 200 mM. In K14Q and K14E cytochrome c-550 the line widths are much smaller at low ionic strength. Wild-type cytochrome c-550 may exist in two exchanging conformations, one of which may represent a more open (non-native) form, in analogy with cytochrome c. However, in the case of cytochrome c-550 this non-native form does not show ligand replacement. The electron self-exchange rates of wild type and mutants have been determined as a function of the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c oxidase

The reactions of bovine cytochrome c oxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis(bipyridine)ruthenium (II) were studied by laser flash photolysis. All of the derivatives form complexes with cytochrome c oxidase at low ionic strength (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in rapid electron transfer to the ferric heme group of cytochrome c, followed by electron transfer to cytochrome c oxidase. The photoreduced heme Fe(II) in the cytochrome c derivative modified at lysine 25 on the periphery of the heme crevice domain transferred an electron to CuA with a rate constant of 1.1 x 10(4) s-1. CuA then transferred an electron to cytochrome a with a rate constant of 2.3 x 10(4) s-1. The derivatives modified at lysines 7, 39, 55, and 60 remote from the heme crevice domain of cytochrome c have nearly the same kinetics. The rate constant for electron transfer from the cytochrome c heme to CuA is greater than 10(5) s-1, and the rate constant for electron transfer from CuA to cytochrome a is 2 x 10(4) s-1. The cytochrome c derivatives modified at lysines 13 and 27 in the heme crevice domain react much more slowly than the other derivatives, with intracomplex rate constants for oxidation of cytochrome c ranging from 1000 to 6000 s-1. The bulky ruthenium group at the heme crevice domain of these derivatives apparently alters the binding orientation, leading to smaller electron-transfer rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamic and kinetic studies of cytochrome c from different species

The direct heterogeneous electron transfer reactions of cytochrome c from different vertebrate species are compared in this report. A kinetic study revealed a biphasic temperature dependence at neutral pH for the formal heterogeneous electron transfer rate constants of these cytochromes. The properties of the electrode-solution interface are believed to cause this biphasic kinetic effect. This effect does not correlate with the conformational transitions evident in both formal potential and circular dichroism (CD) measurements as a function of temperature. The conformational transitions seen in the formal potential and CD measurements arise from the alkaline isomerization and heme crevice changes. These are subtle effects compared with the structural changes associated with unfolding of the protein that are evident in differential scanning calorimetric experiments. The conformational stabilities of cytochrome c between species relate to the differences in their amino acid sequences. The amino acid interactions which stabilize the structure of these different cytochrome c molecules have been evaluated and will be discussed.

Electrochemically induced conformational changes in cytochrome c monitored by Fourier transform infrared difference spectroscopy: influence of temperature, pH, and electrode surfaces.

IR difference spectra between the oxidized and the reduced state of horse heart cytochrome c were obtained for different temperature and pH conditions at various surface-modified electrodes using an optically transparent thin-layer electrochemical cell. These difference spectra reflect changes in protein conformation, side-chain geometries, and protonation upon the redox transition. The IR difference spectra recorded in the 10-40 degrees C temperature range showed thermally induced changes mainly in the amide-I (1700-1600 cm-1) and in the amide-II (ca. 1550 cm-1) spectral regions. Although the position of most of the signals remains unshifted, large differences in their relative amplitude were observed, leading in some cases to the masking and/or the disappearance of some IR signals. In the range 6.8-9.8, increasing pH of the samples led to a decrease in the reduction rate and to spectral changes which closely resemble those obtained by increasing the temperature. Both the thermal and the pH dependence of the reduced-minus-oxidized IR difference spectra reflect the transition of ferricytochrome c from the native to the alkaline form. An analysis of the IR difference spectra shows that the redox transition at neutral pH involves mainly beta-turns and beta-sheet segments of the cytochrome c molecule. However, once the ferricytochrome c alkaline transition is performed, the redox process is coupled to conformational changes involving alpha-helical segments. The shifts in tyrosine vibrational modes observed in the difference spectra obtained at neutral and slightly alkaline pH at high temperatures suggest an intermediate state of the ferricytochrome c in which the heme crevice is more accessible to the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of tyrosine 67 in the cytochrome c heme crevice structure studied by semisynthesis.

Tyr-67 of mitochondrial cytochrome c is thought to be involved in important hydrogen bonding interactions in the hydrophobic heme pocket of the protein (Takano, T., Dickerson, R. E. (1981) J. Mol. Biol. 153:95-115). The role of this highly conserved residue in heme pocket stability was studied by comparing properties of semisynthetic (Phe-67) and (p-F-Phe-67) analogs with those of native cytochrome c and a "control" analog, (Hse-65)cytochrome c. The (Phe-67) and (p-F-Phe-67) analogs have well-developed 695-nm visible absorption bands and are active in a cytochrome c oxidase assay. The reduction potentials of both analogs are lower than the native protein by approximately 50 mV. Although both analogs bind imidazole with higher affinity than the native protein, only the (p-F-Phe-67) analog has a 3- to 5-fold lower binding constant for cyanide. Only the (Phe-67) analog was significantly more stable toward alkaline isomerization. These results are not consistent with stabilization of the native protein heme pocket via hydrogen bonding of Tyr-67 to Met-80. An alternative steric role for Tyr-67 is proposed in which the residue controls the heme reduction potential by limiting the number of internal H2O molecules in the heme pocket.

Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochrome c in the presence of 0.2 M KCl, 0.1 M NaClO4, and 0.2 M KNO3, in the pH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990) J. Protein Chem, 9, 379-387), the change from neutral to low acidic pH's in the presence of salts is a three-step process: state IIIs----state IIIS,a----state IIS----state IS, with pKa's of 3.5 +/- 0.2, 2.2 +/- 0.2, and 1.1 +/- 0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutral pH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State IIIS,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states IIS and IS are also essentially folded forms, but with a smaller degree of protein secondary structure. State IIS has a high-spin hexacoordinated heme iron with a water molecular and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state IS has a high-spin pentacoordinated heme iron, which is about 0.49 A out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state IIIa----state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).

Structural studies of yeast Iso‐1 cytochrome c mutants by resonance Raman spectroscopy

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.

Different functional boundaries for the major antigenic region of two cytochromes c.

The antigenic sites of horse and rat cytochromes c in the major antigenic region were compared using a panel of variant cytochromes c and a large number of BALB/c mouse monoclonal antibodies (mAbs) in competitive ELISAs. The major antigenic region of cytochrome c is located on the surface opposite of that containing the exposed heme crevice. mAbs specific for this region on rat cytochrome c were affected in binding by amino acid substitutions at positions 62 and at one or more of positions 3, 100, 103, and 104 but not by a substitution at position 89. In contrast, mAbs specific for the same region on horse cytochrome c were affected by amino acid substitutions at positions 62, (probably) 60, and 89. Some, but not all, of the anti-horse cytochrome c mAbs were affected by amino acid substitutions at one or more of positions 3, 100, 103, and 104. Thus, the functional boundaries of this antigenic region are different for these two cytochromes c, as shown primarily by the differential effects of residue 89. This distinction is most likely due to one or more of the four amino acid differences between horse and rat cytochromes c on the antigenic surface that result in different physicochemical properties for the horse and rat proteins. The results suggest that as yet unidentified physicochemical parameters, possibly surface topography and/or charge distribution, influence the focusing of antibodies onto the surface of a protein antigen.

Fluorescence study of the conformational properties of myoglobin structure

The porphyrin and tryptophan fluorescence of sperm whale apomyoglobin complexed with protoporphyrin IX has been studied in the pH range 2-13. It has been shown that the fluorescence and absorption spectra of protoporphyrin incorporated into the heme crevice remain constant in the pH range 5.5-10.8 but change significantly at pH less than 5.5 and pH greater than 10.8, due to the acid and alkaline denaturation, respectively, of the complex accompanied by dissociation of protoporphyrin IX. At the same pH ranges, the quantum yield of tryptophanyl fluorescence increases sharply as a result of removal of protoporphyrin, acting as a quencher, from the complex. Other parameters of tryptophanyl fluorescence (maximum position, halfwidth and spectrum shape) change in the alkaline region as well. In the acidic pH range, these parameters change only at pH less than 4.3, indicating that the Trp surroundings are more stable to denaturation than the heme crevice region. Between pH 5.5 and 10.9, where the complex of apomyoglobin with protoporphyrin IX is in its native state, the main parameters of tryptophan fluorescence remain unchanged except for the ratio I325/I350 which diminishes at pH greater than 9.5. Its alteration precedes the alkaline denaturation of the complex and can be explained by a local conformational change induced by the break of the 'salt bridges' essential for the maintenance of the native Mb structure in the N-terminal region. The fluorescence data obtained for apomyoglobin, myoglobin and the complex between protoporphyrin IX and apomyoglobin enable one to compare their structures and to evaluate the role of the porphyrin macrocycle and the iron atom in the formation of the native myoglobin structure and its functioning.

Fluorescence study of the conformational properties of myoglobin structure

The pH-dependent fluorescence of intact sperm whale apomyoglobin (apo-Mb) containing two tryptophans at positions 7 and 14, and of apo-Mb derivatives modified on Trp7 by 2-hydroxy-5-nitrobenzyl bromide (Koshland reagent) and o-nitrophenylsulphenyl chloride, has been studied. The fluorescence of apomyoglobins modified at His residues by iodoacetamide and bromoacetate, and at the N-terminal alpha-NH2 group by methylisothiocyanate, has also been investigated. The individual fluorescent properties of both tryptophans and their contributions to the total spectrum of apo-Mb have been resolved within the pH range 2-12.5. The quantum yield of the 'buried' Trp14 (lambda max at 326 nm) is shown to be twofold higher at pH greater than 8.5 than that of the 'exposed' Trp7 (lambda max at 333 nm). At pH 8.5-5.5 the fluorescence of Trp14 diminished approximately twofold due to quenching by the ionized His residue, most probably His119. The quenching is evidently dynamic because the fluorescence lifetime is shown to be linearly proportional to quantum yield in this pH range. The fluorescence of Trp7 practically does not change between pH 5.5 and 10.0 but increases 2.5-3-fold in the pH range 5.5-4.3 while the contribution of Trp14 remains constant. The conformational changes at the N-terminal and in the region adjacent to it, as well as in the whole apo-Mb molecule in acidic, alkaline and neutral pH ranges, are considered. A relationship is revealed between conformational states of the heme crevice and the N-terminal part of apo-Mb.

Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis

The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance Raman spectroscopic studies of cytochrome c at charged interfaces

The structural changes of cytochrome c bound to charged surfaces have been analyzed by resonance Raman and surface enhanced resonance Raman spectroscopy. These surfaces, which are provided by a silver electrode, heteropolytungstates, and phospholipid vesicles, simulate the binding domain of cytochrome oxidase, the physiological redox partner of cytochrome c. In all model systems, binding of both the oxidated and reduced cytochrome c leads to the formation of two conformational states (I,II). It is shown that the equilibria between these states as well as their individual conformations depend on electrostatic interactions. While state I exhibits the same structure as the uncomplexed cytochrome c, in state II the heme crevice assumes an open structure and the iron-methionine bond is weakened. This structural change is ascribed to the rupture of the Lys-13-Glu-90 salt bridge so that the loop 80–90 swings away from the heme edge and the structure of the heme pocked is loosened. This modification of the heme crevice is accompanied by a negative shift of the redox potential. From the detailed Raman spectroscopic studies of cytochrome c bound to these model systems, characteristic spectral properties of the states I and II are identified. Thus it is possible to analyze the resonance Raman spectra of the cytochrome c/ cytochrome oxidase complex, where the state II can also be detected. These findings suggest that the opening of the heme crevice plays a functional role for the physiological electron transfer process.

Changing the invariant proline-30 of rat and Drosophila melanogaster cytochromes c to alanine or valine destabilizes the heme crevice more than the overall conformation.

Drosophila melanogaster and rat cytochromes c in which proline-30 was converted to alanine or valine were expressed in a strain of baker's yeast, Saccharomyces cerevisiae, where they sustained aerobic growth. The mutations had no significant effect on the spectra or redox potentials but altered drastically the stability of the bond between the methionine-80 sulfur and the heme iron, as judged by four criteria: (i) the alkaline pKa values of the 695-nm band of the ferric form of the mutant proteins decreased by almost 1 pH unit as compared to the wild types; (ii) the acid pKa values increased by 0.5 to 1.2 pH units; (iii) the 695-nm band half-disappeared at temperatures 10-20 degrees C lower in the mutant proteins than in the wild types; and (iv) the 695-nm band of the mutant proteins was susceptible to concentrations of urea that had little influence on their overall structure. The valine-substituted rat cytochrome c had properties intermediate between those of the wild type and the alanine mutant. The destabilized coordinative bond is located in space a long distance from the mutation site. It is suggested that the mutations weaken the hydrogen bond between the carbonyl of residue 30 and the imino group of the imidazole of histidine-18, modifying the bonding of the heme iron by that imidazole, which, in turn, through a trans effect, weakens the bond between the heme iron and the other axial ligand, the sulfur of methionine-80. Alternatively, the effect of the mutations may be propagated allosterically along the peptide chain.

Polyanion binding to cytochrome c probed by resonance Raman spectroscopy

The interaction of ferricytochrome c with negatively charged heteropolytungstates was studied by resonance Raman spectroscopy. In analogy to previous findings on ferricytochrome c bound to other types of charged interface (Hildebrandt, P. and Stockburger, M. (1989) Biochemistry 28, 6710–6721, 6722–6728), it was shown that in these complexes the conformational states I and II are stabilized. While in state I, the structure is the same as is in the uncomplexed heme protein, in state II three different coordination configurations coexist, i.e., a six-coordinated low-spin, a five-coordinated high-spin and a six-coordinated high-spin form. These configurations constitute thermal coordination equilibria whose thermodynamic properties were determined. The detailed analysis of the low-frequency resonance Raman spectra reveals that in state II the heme pocket assumes an open structure leading to a significantly higher flexibility of the heme group compared to the native ferricytochrome c. It is concluded that these structural changes are the result of Coulombic attractions between the polyanions and the lysine residues around the exposed heme edge which destabilize the heme crevice. Modifications of these interactions upon variation of the ionic strength, the pH or the type of the polytungstate are sensitively reflected by changes of the coordination equilibria in state II as well as of the conformational equilibrium of state I and state II. The conformational changes in state II significantly differ from those associated with the alkaline transition of ferricytochrome c. However, there are some structural similarities between the acid form of the heme protein stable below pH 2.5 in aqueous solution and the six-coordinated high-spin configuration of the bound ferricytochrome c at neutral pH (state II). This suggests that electrostratic interactions with the heteropolytungstates perturb the ionic equilibria of those amino acid side chains which are involved in the acid-induced transition leading to a significant upshift of the apparent p K a.

Horse heart ferricytochromec: Conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochrome c in the pH range 6-0.8 and at the lowest possible ionic strengths, in water, and at 30 degrees C are reported. The neutral pH form, state III, changes to the acidic pH form, state I, through a three-step process: state III in equilibrium with IIIa in equilibrium with state II in equilibrium with state I, with pKa's of 3.6 +/- 0.3, 2.7 +/- 0.2, and 1.2 +/- 0.2, depending on the monitoring probe, respectively. State IIIa ferricytochrome c is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochrome c is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin, and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 A.

High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c

The structure of yeast iso-1-cytochrome c has been refined against X-ray diffraction data to a nominal resolution of 1.23A˚. The atomic model contains 893 protein atoms, as well as 116 water molecules and one sulfate anion. Also included in the refinement are 886 hydrogen atoms belonging to the protein molecule. The crystallographic R-factor is 0.192 for the 12, 513 reflections with F ⩾ 3σ( F) in the resolution range 6.0 to 1.23A˚. Co-ordinate accuracy is estimated to be better than 0.18A˚. The iso-1-cytochrome c molecule has the typical cytochrome c fold, with the polypeptide chain organized into a series of α-helices and reverse turns that serve to envelop the heme prosthetic group in a hydrophobic pocket. Inspection of the conformations of helices in the molecule shows that the local environments of the helices, in particular the presence of intrahelical threonine residues, cause distortions from ideal α-helical geometry. Analysis of the internal mobility of iso-1-cytochrome c, based on refined crystallographic temperature factors, shows that the most rigid parts of the molecule are those that are closely associated with the heme group. The degree of saturation of hydrogen-bonding potential is high, with 90% of all polar atoms found to participate in hydrogen bonding. The geometry of intramolecular hydrogen bonds is typical of that observed in other high-resolution protein structures. The 116 water molecules present in the model represent about 41% of those expected to be present in the asymmetric unit. The majority of the water molecules are organized into a small number of hydrogen-bonding networks that are anchored to the protein surface. Comparison of the structure of yeast iso-1-cytochrome c with those of tuna and rice cytochromes c shows that these three molecules have very high structural similarity, with the atomic packing in the heme crevice region being particularly highly conserved. Large conformational differences that are observed between these cytochromes c can be explained by amino acid substitutions. Additional subtle differences in the positioning of the side-chains of several highly conserved residues are also observed and occur due to unique features in the local environments of each cytochrome c molecule. Considering the structures of reduced yeast iso-1- and tuna cytochromes c, and of oxidized tuna and rice cytochromes c, oxidation state-dependent conformational changes are observed to occur in the vicinity of a buried water molecule on the Met80 side of the heme pocket, confirming results obtained previously by T. Takano and R. E. Dickerson.

Photoinduced electron transfer within complexes between plastocyanin and ruthenium bisbipyridine dicarboxybipyridine cytochrome c derivatives

A new technique has been developed to measure intracomplex electron transfer between cytochrome c and its redox partners. Cytochrome c derivatives labeled at single lysine amino groups with ruthenium bisbipyridine dicarboxybipyridine were prepared as previously described [Pan, L.P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Excitation of RuII with a short light pulse resulted in the formation of the excited-state RuII*, which rapidly transferred an electron to the ferric heme group to form FeII and RuIII. Aniline was included in the buffer to reduce RuIII to RuII, leaving the heme group in the ferrous state. This process was complete within the lifetime of the light pulse. When plastocyanin was present in the solution, electron transfer from the ferrous heme of cytochrome c to CuII in plastocyanin was observed. All of the ruthenium cytochrome c derivatives formed electrostatic complexes with plastocyanin at low ionic strength, allowing intracomplex electron-transfer rate constants to be measured. The rate constants for derivatives modified at the indicated lysines were as follows: Lys 13, 1920 s-1; Lys 8, 1480 s-1; Lys 7, 1340 s-1; Lys 86, 1020 s-1; Lys 25, 820 s-1; Lys 72, 800 s-1; Lys 27, 530 s-1. It is interesting that the derivative modified at lysine 13 at the top of the heme crevice had the largest rate constant, while lysine 27 at the right side of the heme crevice had the smallest.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational changes in cytochrome c and cytochrome oxidase upon complex formation: a resonance Raman study

The fully oxidized complex of cytochrome c and cytochrome oxidase formed at low ionic strength was studied by resonance Raman spectroscopy. The spectra of the complex and of the individual components were compared over a wide frequency range using Soret band excitation. In both partners of the complex, structural changes occur in the heme groups and in their immediate protein environment. The spectra of the complex in the 1600-1700 cm-1 frequency range were dominated by bands from the cytochrome oxidase component, whereas those in the 300-500 cm-1 range were dominated by bands from the cytochrome c component, hence allowing separation of the contributions from the two individual species. For cytochrome c, spectral changes were observed which correspond to the induction of the conformational state I and the six-coordinated low-spin configuration of state II on binding to cytochrome oxidase. While in state I the structure of cytochrome c is essentially the same as in solution, state II is characterized by a structural rearrangement of the heme pocket, leading to a weakening of the axial iron-methionine bond and an opening of the heme crevice which is situated in the center of the binding domain for cytochrome oxidase. The relative contributions of the two cytochrome c states were estimated to be approximately in the ratio 1:1 in the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

A polypeptide chain-refolding event occurs in the Gly82 variant of yeast iso-1-cytochrome c

The replacement of Phe82 in yeast iso-1-cytochrome c by a glycine residue substantially alters both the tertiary structure and electron transfer properties of this protein. The largest structural change involves a polypeptide chain refolding of residues 79 through 85. Refolding places glycines 82, 83 and 84 immediately adjacent to the plane of the heme group in a spatial positioning comparable to that of the phenyl ring of Phe82 in the wild-type protein. Despite this perturbation in structure, solvent accessibility computations show that heme solvent exposure has not increased in the Gly82 variant protein. However, refolding does result in the introduction of a number of polar groups into the hydrophobic heme pocket. This appears to be responsible for the decreased reduction potential of the heme in this protein. The present study, along with that of the Ser82 variant protein (Louie et al., 1988 b), clearly establishes the link between dielectric constant within the heme crevice and reduction potential. The further anomalously low electron transfer activity of the Gly82 variant protein would appear to arise from two factors. First, the polypeptide chain medium now adjacent to the heme is unable to facilitate electron transfer in a manner similar to that of the aromatic side-chain of Phe82. Second, polypeptide chain refolding significantly alters the surface contour of the Gly82 protein rendering it less suitable to interact with the corresponding complementary surfaces of redox partners. Our data support the conclusion that Phe82 plays a number of roles in the electron transfer process mediated by yeast iso-1-cytochrome c. These include the maintenance of the heme environment, provision of an optimal medium along the path of electron transfer and formation of interactions at the contact interface in complexes with redox partners.