Cyanide binding to the cytochrome c ferric heme octapeptide. A model for anion binding to the active site of high spin ferric heme proteins

Equilibrium constants for the binding of cyanide to the ferric heme c octapeptide in 20% ethylene glycol, 50% buffer were measured spectrophotometrically. The equilibrium constant for cyanide binding at 20 degrees C and pH 7.4 is 3.47 X 10(7), which is approximately 15-fold lower than that observed for cyanide binding to methemoglobin or metmyoglobin. Equilibrium constants at several temperatures exhibited an apparent van't Hoff relationship, yielding thermodynamic values of delta H degrees = -79,000 J/mol (-18,900 cal/mol) and delta S degrees = J/degrees K mol (-30.1 e.u.). Comparison of the ratio of equilibrium constants for cyanide ligation to methemoglobin the heme octapeptide with the ratio of equilibrium constants for azide ligation to methemoglobin and the heme octapeptide suggests that cyanide binding to the methemoproteins is much smaller than expected by comparison to azide binding. The differences in the ratios, the thermodynamic values, and the preferred binding geometries suggest that CN- ligation, like CO ligation, is sterically hindered. A comparison of these ratios to similar ratios for CO, O2, and NO binding suggests that the Fe-CN bond angle is less subject to distortion than the Fe-CO bond and/or additional binding interactions contribute to N3- but not to CN-binding to the protein.     

Azide binding to the cytochrome c ferric heme octapeptide. A model for anion binding to the active site of high spin ferric heme proteins.

Equilibrium constants for the binding of azide to the ferric heme c octapeptide in 50% ethylene glycol 50% buffer were measured spectrophotometrically. The equilibrium constant for azide binding at 20 degrees C and pH* 7.4 is 29.2, which is approximately 3 to 4 orders of magnitude lower than that observed for azide binding to various ferric hemeproteins. The equilibrium constant was indepent of pH* in the range from 7 to 8. Equilibrium constants at several temperatures exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta H0 = -26,100 J/mol (-6240 cal/mol) and delta S0 = -61.5 J/0K mol (-14.7 e.u.). Comparison of these values to the values for the heme proteins enables one to explain the differences in equiliberium constants in terms of differences in the polarity of the heme environments. The results are consistent with the concept that the oxygen affinity of heme complexes increases with the polarity of the heme environment. The data also suggest that an increase in the polarity of the heme environment should result in a corresponding increase in the susceptibility of ferrous heme dioxygen complexes toward oxidation by the dioxygen.     

The effect of temperature on the spectroscopic properties of the heme c octapeptide from cytochrome c.

The effect of temperature on the optical properties of the acetylated heme c octapeptide from cytochrome c was examined. At above ambient temperatures the observed optical spectrum with maxima at 549 and 424 nm was characteristic of high-spin ferrous hemeproteins. At below ambient temperatures the optical spectrum became characteristic of low-spin ferrous hemeproteins with maxima at 547, 518, and 410 nm. A thermodynamic characterization of this two component system yielded a deltaHO of -10.1 +/- 0.7 kcal/mol and a delta S0 of-37.6 +/- 2.5 e.u. for the temperature dependent process. Discussion of the spectroscopic and thermodynamic parameters was presented in terms of the consistent magnetic and structural properties of heme complexes.     

Studies on the heme environment of horse heart ferric cytochrome c. Azide and imidazole complexes of ferric cytochrome c.

Horse heart ferric cytochrome c was investigated by the following three methods: (I) Light absorption spectrophotometry at 23 degrees C and 77 degrees K; (II) Electron paramagnetic resonance (EPR) spectroscopy at 20 degrees K; (III) Precise equilibrium measurements of ferric cytochrome c with azide and imidazole between 14.43 and 30.90 degrees C. I and II have demonstrated that: (1) Ferric cytochrome c azide and imidazole complexes were in the purely low spin state between 20 degrees K and 23 degrees C; (2) The energy for the three t2g orbitals calculated in one hole formalism shows that azide or imidazole bind to the heme iron in a similar manner to met-hemoglobin azide or imidazole complexes, respectively. III has demonstrated that: (1) The change of standard enthalpy and that of standard entropy were -2.3 kcal/mol and -1.6 cal/mol per degree for the azide complex formation, and -1.4 kcal/mol and 2.9 cal/mol per degree for the imidazole complex formation. (2) A linear relationship between the change of entropy and that of enthalpy was observed for the above data for the cyanide complex formation. The complex formation of ferric cytochrome c was discussed based on the results of X-ray crystallographic studies compared with hemoglobin and myoglobin.     

Nuclear magnetic resonance studies of hemoproteins. VIII. Proton NMR studies of the binding of various nitrogenous ligands to ferric cytochrome c.

The structure of the heme environment of horse heart ferric cytochrome c was examined in the presence of various nitrogenous bases at several temperatures with the aid of hyperfine shifted proton NMR spectra at 220 MHz. The resonance positions and line widths of the signals for the peripheral methyl groups of the heme exhibited distinctive features of its low-spin state characteristic of each external ligand. In the imidazole complex of ferric cytochrome c, remarkable line sharpening of the heme-linked proton signals was encountered on raising the temperature. This may be related to the apoprotein perturbation on the binding of external ligand to the heme iron. These spectral peculiarities were discussed in relation to the electronic structure of the heme, the basicity of the external ligand and the van der Waals contact interaction between heme side chains and apoprotein.     

Nuclear magnetic resonance studies of hemoproteins: VIII. Proton NMR studies of the binding of various nitrogenous ligands to ferric cytochrome c

The structure of the heme environment of horse heart ferric cytochrome c was examined in the presence of various nitrogenous bases at several temperatures with the aid of hyperfine shifted proton NMR spectra at 220 MHz. The resonance positions and line widths of the signals for the peripheral methyl groups of the heme exhibited distinctive features of its low-spin state characteristic of each external ligand.In the imidazole complex of ferric cytochrome c, remarkable line sharpening of the heme-linked proton signals was encountered on raising the temperature. This may be related to the apoprotein perturbation on the binding of external ligand to the heme iron. These spectral peculiarities were discussed in relation to the electronic structure of the heme, the basicity of the external ligand and the van der Waals contact interaction between heme side chains and apoprotein.     

A trypsin-resistant heme peptide from cardiac cytochrome c1.

A tryptic resistant heme peptide has been prepared and purified from cardiac cytochrome c1. This purified peptide is not further hydrolyzed by reactions of other proteolytic enzymes, such as pronase. The peptide contains 2 residues each of serine, cysteine and valine, and 1 residue each of alanine, methionine, tyrosine, histidine, arginine, proline, glutamic acid (glutamine) and aspartic acid. The intensity of the absorption spectrum of the peptide has been found to be dependent upon, but the positions of the absorption maxima do not vary with, concentration. The heme peptide does not show multiple splitting of absorption peaks at liquid N2 temperatures as does the intact cytochrome C1. However, cyanide rapidly reacts with the peptide and causes significant spectral changes. CD spectra of the peptide exhibit a typical profile of a non-structured heme peptide with positive CD bands in the Soret region and around 250 nm, and a broad negative extreme of 320-360 nm. The similarities and differences between the tryptic resistant heme peptides from cytochromes c1 and c have been compared.     

Yeast cytochrome c peroxidase. Coordination and spin states of heme prosthetic group

Electronic absorption and electron paramagnetic resonance (EPR) spectroscopic examinations revealed that a freshly prepared cytochrome c peroxidase (CCP) contains a penta-coordinated high spin ferric protoheme group. The penta-coordinated high spin state of fresh CCP is maintained in a remarkably wide range of pH (4-8). The freezing of fresh CCP induces the reversible coordination of an internal strong field ligand to the heme iron to form a hexa-coordinated low spin compound, which shows EPR extrema at gx = 2.70, gy = 2.20 and gz = 1.78. In the presence of glycerol the freezing-induced artifacts are eliminated and the fresh enzyme exhibits an EPR spectrum of rhombically distorted axial symmetry with EPR extrema at gx = 6.4, gy = 5.3, and gz = 1.97 at 10 K, characteristic of the penta-coordinated high spin enzyme. Upon aging CCP is converted to a hexa-coordinated high spin state due to the coordination of an internal weak field ligand to the heme iron. This conversion is accelerated at acidic pH values, and its reversibility varies from fully reversible to irreversible depending on the degree of enzyme aging. The aging-induced hexa-coordinated CCP is unreactive with hydrogen peroxide and exhibits an EPR spectrum of purely axial symmetry with extrema at g = 6 and g = 2 and an electronic absorption spectrum with an intensified Soret band at 408 nm (epsilon 408 nm = 120 mM-1 cm-1) and a blue-shifted charge-transfer band at 620 nm. Spectroscopic properties of different coordination and spin states of fresh and aged CCPs are compiled in order to formulate a generalized spectroscopic characterization of penta- and hexa-coordinated high spin ferric hemoproteins.     

ENDOR from nitrogens and protons in low spin ferric heme and hemoprotein.

Electron nuclear double resonance (ENDOR) signals have been obtained from iron-linked nitrogens in frozen solutions of cytochrome c, metmyoglobin cyanide, and a low spin protohemin mercaptide complex. Hyperfine couplings from heme protons have also been obtained from metmyoglobin cyanide and from a low spin protohemin cyanide complex. Several of these proton resonances are assigned to specific heme protons.     

Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor

Background

For many pathogenic microorganisms, iron acquisition represents a significant stress during the colonization of a mammalian host. Heme is the single most abundant source of soluble iron in this environment. While the importance of iron assimilation for nearly all organisms is clear, the mechanisms by which heme is acquired and utilized by many bacterial pathogens, even those most commonly found at sites of infection, remain poorly understood.

Methods

An alternative protocol for the production and purification of the outer membrane hemoglobin receptor (HmbR) from the pathogen Neisseria meningitidis has facilitated a biophysical characterization of this outer membrane transporter by electronic absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman techniques.

Results

HmbR co-purifies with 5-coordinate high spin ferric heme bound. The heme binding site accommodates exogenous imidazole as a sixth ligand, which results in a 6-coordinate, low-spin ferric species. Both the 5- and 6-coordinate complexes are reduced by sodium hydrosulfite. Four HmbR variants with a modest decrease in binding efficiency for heme have been identified (H87C, H280A, Y282A, and Y456C). These findings are consistent with an emerging paradigm wherein the ferric iron center of bound heme is coordinated by a tyrosine ligand.

Conclusion

In summary, this study provides the first spectroscopic characterization for any heme or iron transporter in Neisseria meningitidis, and suggests a coordination environment heretofore unobserved in a TonB-dependent hemin transporter.

General Significance

A detailed understanding of the nutrient acquisition pathways in common pathogens such as N. meningitidis provides a foundation for new antimicrobial strategies.     

Perturbation of apoptosis upon binding of tRNA to the heme domain of cytochrome c

In response to apoptotic stimuli, cytochrome c, an inter-membrane space protein is released from mitochondria to activate the cascade of caspases that leads to apoptosis. Recent evidence suggests that cytochrome c interacts with tRNA in the cytoplasm and this interaction was shown to inhibit the caspase mediated apoptotic process. Interestingly, cytochrome c does not contain any putative RNA binding domain. In this report, we sought to define the structural component of cytochrome c that is involved in binding of tRNA. By using gel mobility shift assays, we show that holocytochrome c can interact with tRNA but not apocytochrome c that lacks the heme domain suggesting that heme is essential for the interaction of cytochrome c to tRNA. In addition, using in vitro cross linking and circular dichroism spectroscopic studies, we show that cytochrome c can undergo heme mediated oligomerization. Prevention of heme mediated oligomerization of cytochrome c by potassium ferricyanide treatment prevents the binding of tRNA and promotes caspase activation. Our studies provide a novel regulation of apoptosis by heme dependent tRNA interaction to cytochrome c.     

Isocyanide binding kinetics to monomeric hemoproteins. A study on the ligand partition between solvent and heme pocket.

The kinetics of methyl-, ethyl-, iso-propyl-, and ter-butyl-isocyanide binding to Aplysia limacina myoglobin (distal His----Lys) and the isolated beta chains from hemoglobin Zurich (distal His----Arg) have been investigated by flash photolysis at various temperatures above 0 degrees C. Sperm whale (Physter catodon) myoglobin and the isolated beta chains from normal adult hemoglobin have been used as references. In most reaction systems investigated the apparent extent of photolysis increases with temperature. For sperm whale myoglobin and the normal beta chains the increase is of the same magnitude and not correlated to the type of ligand used. On the contrary, for the two proteins lacking the distal histidine, the phenomenon is dependent on the size of the alkyl side chain of the ligand. The results, analyzed on the basis of the multibarrier model (Austin, R.H., K.W. Beeson, L. Eisenstein, H. Frauenfelder, and I.C. Gunsalus, 1975, Biochemistry, 16:5355-5373), suggest that the partition of the ligand molecules between the solvent and the heme pocket, occurring during the photolysis process, is primarily determined by interactions between the ligand and residues in the heme cavity rather than by diffusion through the protein matrix.     

Synergistic anion and metal binding to the ferric ion-binding protein from Neisseria gonorrhoeae

The 34-kDa periplasmic iron-transport protein (FBP) from Neisseria gonorrhoeae (nFBP) contains Fe(III) and (hydrogen)phosphate (synergistic anion). It has a characteristic ligand-to-metal charge-transfer absorption band at 481 nm. Phosphate can be displaced by (bi)carbonate to give Fe.CO(3).nFBP (lambda(max) 459 nm). The local structures of native Fe-PO(4)-nFBP and Fe.CO(3).nFBP were determined by EXAFS at the FeK edge using full multiple scattering analysis. The EXAFS analysis reveals that both phosphate and carbonate ligands bind to FBP in monodentate mode in contrast to transferrins, which bind carbonate in bidentate mode. The EXAFS analysis also suggests an alternative to the crystallographically determined position of the Glu ligand, and this in turn suggests that an H-bonding network may help to stabilize monodentate binding of the synergistic anion. The anions oxalate, pyrophosphate, and nitrilotriacetate also appear to serve as synergistic anions but not sulfate or perchlorate. The oxidation of Fe(II) in the presence of nFBP led to a weak Fe(III).nFBP complex (lambda(max) 471 nm). Iron and phosphate can be removed from FBP at low pH (pH 4.5) in the presence of a large excess of citrate. Apo-FBP is less soluble and less stable than Fe.nFBP and binds relatively weakly to Ga(III) and Bi(III) but not to Co(III) ions, all of which bind strongly to apo-human serum transferrin.     

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase.

Dilatometry is a sensitive technique for measuring volume changes occurring during a chemical reaction. We applied it to the reduction-oxidation cycle of cytochrome c oxidase, and to the binding of cytochrome c to the oxidase. We measured the volume changes that occur during the interconversion of oxidase intermediates. The numerical values of these volume changes have allowed the construction of a thermodynamic cycle that includes many of the redox intermediates. The system volume for each of the intermediates is different. We suggest that these differences arise by two mechanisms that are not mutually exclusive: intermediates in the catalytic cycle could be hydrated to different extents, and/or small voids in the protein could open and close. Based on our experience with osmotic stress, we believe that at least a portion of the volume changes represent the obligatory movement of solvent into and out of the oxidase during the combined electron and proton transfer process. The volume changes associated with the binding of cytochrome c to cytochrome c oxidase have been studied as a function of the redox state of the two proteins. The volume changes determined by dilatometry are large and negative. The data indicate quite clearly that there are structural alterations in the two proteins that occur on complex formation.