Mutagenic, electrochemical, and crystallographic investigation of the cytochrome b5 oxidation-reduction equilibrium: involvement of asparagine-57, serine-64, and heme propionate-7

A gene coding for lipase-solubilized bovine liver microsomal cytochrome b5 has been synthesized, expressed in Escherichia coli, and mutated at functionally critical residues. Characterization of the recombinant protein revealed that it has a reduction potential that is approximately 17 mV lower than that of authentic wild-type protein at pH 7 (25 degrees C). Structural studies determined that the recombinant protein differed in sequence from authentic wild-type cytochrome b5 owing to three errors in amidation status in the published sequence for the protein on which the gene synthesis was based. The structural origin of the lower reduction potential exhibited by the triple mutant has been investigated through X-ray crystallographic determination of the three-dimensional structure of this protein and is attributed to the presence of Asp-57 within 3.3 A of heme vinyl-4 in the mutant. In addition, the model developed by Argos and Mathews [Argos, P., & Mathews, F.S. (1975) J. Biol. Chem. 250, 747] for the change in cytochrome b5 oxidation state has been studied through mutation of Ser-64 to Ala. In this model, Ser-64 is postulated to stabilize the oxidized protein through H-bonding interactions with heme propionate-7 that orients this propionate group 6.2 A from the heme iron. Spectroelectrochemical studies of a mutant in which Ser-64 has been changed to an alanyl residue demonstrate that this protein has a reduction potential that is 7 mV lower than that of the wild-type protein; moreover, conversion of the heme propionate groups to the corresponding methyl esters increases the potential by 67 mV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c.

The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR. The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein. In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved. Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38. The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3188-3197] appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification.     

Structural basis for the variation of pH-dependent redox potentials of Pseudomonas cytochromes c-551

The redox potentials of many c-type cytochromes vary with pH over the physiological pH range. We have investigated the pH dependence of redox potential for the four homologous cytochromes c-551 from Pseudomonas aeruginosa, Pseudomonas stutzeri strain 221, Pseudomonas stutzeri strain 224, and Pseudomonas mendocina . The pH dependence is due to an ionizable group that ionizes with pKox in ferricytochrome c-551 but with a higher pK, pKred , in ferrocytochrome c-551. For P. aeruginosa cytochrome c-551 it has been shown that this ionizable group is one of the heme propionic acid substituents [Moore, G. R., Pettigrew , G. W., Pitt , R. C., & Williams, R. J. P. (1980) Biochim. Biophys. Acta 590, 261-271]but the values of pKox and pKred are significantly lower in this protein than in the other three cytochromes. NMR and chemical modification studies show that for the two P. stutzeri cytochromes c-551 and P. mendocina cytochrome c-551, this propionic acid substituent is again important for the pH dependence of the redox potential. However, a histidine occurring at position 47 in their sequences hydrogen bonds to the propionic acid and thereby raises its pK. In P. aeruginosa cytochrome c-551, His-47 is substituted by Arg-47. Hydrogen-bonding schemes involving His-47 and the propionic acid are proposed.     

Conformation of cobrotoxin in aqueous solution as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His-32 is slightly lower than the pKa value (6.65) of the reference model of N-acetylhistidine methylamide, because of the electrostatic interaction with Arg-33 and Asp-31. The pKa value (5.3--5.4) of His-4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg-59. The hydrogen-deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole-bearing models. The second-order rate constants of N-acetylhistidine methylamide, N-acetylhistidine and imidazole acetic acid satisfy the Br?nsted relation. With reference to this Br?nsted relation, the imidazole ring of His-32 is confirmed to be exposed. The imidazole ring of His-4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg-59 in the proximity. All the methyl proton resonances are assigned to amino-acid types, by conventional double-resonance method and more effectively by the spin-echo double-resonance method. Eight methyl proton resonances are identified as due to the gamma and/or delta-methyl groups of Val-46, Leu-1, Ile-50 and Ile-52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nulcear Overhauser effect enhancements. The aromatic proton resonances of Trp-29 are affected by the ionizable groups of Asp-31, His-32 and Tyr-35. The methyl groups of Ile-50 are in the proximity to the aromatic ring of Trp-29 and the methyl groups of Ile-52 are in the proximity to Tyr-25. The highest-field methyl proton resonance is due to a threonine residue in the proximity to His-4. The appreciable temperature-dependent chemical shift of this methyl proton resonance suggests a temperature-dependent local conformational equilibrium around the His-4 residue of the first loop of the cobrotoxin molecule.     

Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides.

The complete primary structure of an unusual soluble cytochrome c isolated from the obligate methylotrophic bacterium Methylophilus methylotrophus has been determined to contain 124 amino acids and to have an average molecular mass of 14293.0 Da. The sequence has two unusual features: firstly, the location of the heme-binding cysteines is far downstream from the N-terminus, namely at positions 49 and 52; secondly, an extra pair of cysteine residues is present near the C-terminus. In both respects, cytochrome c" is similar to the oxygen-binding heme protein SHP from the purple phototrophic bacterium Rhodobacter sphaeroides. In contrast to SHP, cytochrome c" changes from low-spin to high-spin upon reduction, due to dissociation of a sixth heme ligand histidine which is identified as His-95 by analogy to the class I cytochromes c. The distance of His-95 from the heme (41 residues) and the presence of certain consensus residues suggests that cytochrome c" is the second example of a variant class I cytochrome c.     

Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and 1H NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E0') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pKa values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S0'rc and Delta H0'rc) for the various protein conformers [low- and high-T native (N1 and N2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees ' values very similar to those of the mammalian proteins; (ii) the N1 --> N2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7. 5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pKa for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c2.     

Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis.

The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.     

NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans.

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax approximately 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax approximately 3.5 converts mainly to a signal at g approximately 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.     

Proton NMR assignments of heme contacts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects.

Proton NMR assignments of the heme pocket and catalytically relevant amino acid protons have been accomplished for cyanide-ligated yeast cytochrome c peroxidase. This form of the protein, while not enzymatically active itself, is the best model available (that displays a resolvable proton NMR spectrum) for the six-coordinate low-spin active intermediates, compounds I and II. The assignments were made with a combination of one- and two-dimensional nuclear Overhauser effect methods and demonstrate the utility of NOESY experiments for paramagnetic proteins of relatively large size (Mr 34,000). Assignments of both isotope exchangeable and nonexchangeable proton resonances were obtained by using enzyme preparations in both 90% H2O/10% D2O and, separately, in 99.9% D2O solvent systems. Complete resonance assignments have been achieved for the proximal histidine, His-175, and His-52, which is a member of the catalytic triad on the distal side of the heme. In addition, partial assignments are reported for Trp-51 and Arg-48, catalytically important residues, both on the distal side. Aside from His-175, partial assignments for amino acids on the proximal side of the heme are proposed for the alanines at primary sequence positions 174 and 176 and for Thr-180 and Leu-232.     

Ligand binding and structural perturbations in cytochrome c peroxidase. A crystallographic study

Crystal structures of the complexes formed between cytochrome c peroxidase and cyanide, nitric oxide, carbon monoxide, and fluoride have been determined and refined to 1.85 A. In all four complexes significant changes occur in the distal heme pocket due to movement of Arg-48, His-52, and a rearrangement of active site water molecules. In the cyanide, nitric oxide, and carbon monoxide complexes, Arg-48 moves away from the ligand while in the fluoride complex Arg-48 moves in toward the ligand to form a hydrogen bond or ion pair with the fluoride. More subtle changes occur on the proximal side of the heme. In an earlier study at lower resolution (Edwards, S. L., Kraut, J., and Poulos, T. L. (1988) Biochemistry 27, 8074-8081), we found that nitric oxide binding causes perturbations in the proximal domain involving Trp-191 which has been confirmed by the present study. Trp-191 is stacked parallel to and in contact with the proximal ligand, His-175. Nitric oxide binding results in a slight movement of Trp-191 away from His-175 and a large increase in crystallographic temperature factors indicating increased mobility of these residues on the proximal side of the heme. These proximal-side changes are unique to nitric oxide and are not related strictly to spin-state or oxidation state of the iron atom since similar changes were not observed in the cyanide (low-spin ferric), carbon monoxide (low-spin ferrous), or fluoride (high-spin ferric) complexes.     

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.     

Effect of active site and surface mutations on the reduction potential of yeast cytochrome c peroxidase and spectroscopic properties of the oxidized and reduced enzyme

The reduction potentials of 22 yeast cytochrome c peroxidase (CcP) mutants were determined at pH 7.0 in order to determine the effect of both heme pocket and surface mutations on the Fe(III)/Fe(II) redox couple of CcP, as well as to determine the range in redox potentials that could be obtained through point mutations in the enzyme. Spectroscopic properties of the Fe(III) and Fe(II) forms of the mutant enzymes are also reported. The mutations include variants in the distal and proximal heme pockets as well as on the enzyme surface and involve single, double, and triple point mutations. A spectrochemical redox titration technique used in this study gave an E(0') value of -189 mV for yeast CcP compared to a previously reported value of -194 mV determined by potentiometry [C.W. Conroy, P. Tyma, P.H. Daum, J.E. Erman, Biochim. Biophys. Acta 537 (1978) 62-69]. Both positive and negative shifts in the reduction potential from that of the wild-type enzyme were observed, spanning a range of 113 mV. The His-52-->Asn mutation gave the most negative potential, -259 mV, while a triple mutant in which the three distal pocket residues, Arg-48, Trp-51, and His-52, were all converted to leucine residues gave the most positive potential, -146 mV.     

Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase.

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.     

Structural consequences of b- to c-type heme conversion in oxidized Escherichia coli cytochrome b562

An NMR characterization of the 98Arg --> Cys variant of iron (III)-containing cytochrome b562 from Escherichia coli has been performed and the solution structure obtained. This variant has a covalent bond between the heme and Cys 98, thus mimicking the heme binding in cytochrome c. The R98C cytochrome is shown to have a significantly increased stability, compared to that of wild type, toward thermal and chemical denaturation. In water at 20 degrees C it is 5.60 kJ mol-1 more stable than the WT protein, measured by equilibrium guanidine hydrochloride denaturation. The structure has been obtained through two-dimensional total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments and through three-dimensional NOESY-15N heteronuclear multiple quantum coherence (HMQC). By these methods, 85% of protons and 100% of backbone nitrogens were assigned. 2145 meaningful nuclear Overhauser effects (NOEs) (20 NOEs per residue), 45 backbone 3J values, and 397 pseudocontact shifts were used to obtain a family of 35 members, which were then energy-minimized. The root-mean-square deviation (RMSD) with respect to the average structure is 0.50 +/- 0.07 for the backbone and 1.01 +/- 0.08 for the heavy atoms. The magnetic anisotropy resulting from analysis of the pseudocontact shifts indicates an anisotropy that is an intermediate between that of the wild-type, which is the smallest, and cytochrome c. The g values confirm a higher anisotropy of the variant with respect to the wild-type protein. The chirality of the heme 2 alpha carbon is the same as that in all naturally occurring cytochromes c. The overall secondary structure and tertiary structure are very similar to the wild type. The removal of Arg 98 causes a change in the pH-dependent properties. The pKa, proposed to be due to deprotonation of the coordinated histidine, is 1.5 units higher than in the wild type, consistent with the lack of the positive charge of Arg 98 close to the ionizable group. This is further support for the coordinated histidine being the titratable group with an alkaline pKa in the wild-type protein. The pattern of the shifts of the heme methyl groups is different than in the wild-type protein, presumably due to alteration of the electronic structure by the presence of the covalent bond between the protein and the heme. The difference in stability between the variant and wild-type protein is discussed in terms of the structural information.     

Spectroelectrochemical study of cytochrome c oxidase: pH and temperature dependences of the cytochrome potentials. Characterization of site-site interactions

The cytochrome a and a3 sites in uninhibited, detergent-solubilized cytochrome c oxidase have been studied under a wide range of conditions using thin-layer spectroelectrochemistry. The observed absorbance changes at the alpha and Soret absorbance maxima have been used together to estimate the extents of reduction of cytochromes a and a3, using the absorbance properties of these cytochromes deduced from previous measurements employing ligand inhibition of cytochrome a3. The resulting Nernst plots, combined with the results of parallel studies on the carbon monoxide-inhibited enzyme (Ellis, W. R., Jr., Wang, H., Blair, D. F., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 161-167; Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 167-171), indicate that the cytochrome a site participates in anticooperative thermodynamic interactions which involve all three of the other metal sites in the protein. Using an analysis which resolves the intrinsic thermodynamic properties of the cytochromes from the effects of the intersite interactions, the pH, temperature, and ionic strength dependences of the cytochrome reduction potentials have been measured. The standard entropy of reduction of cytochrome a in the native enzyme is large and negative, in agreement with measurements on the carbon monoxide-inhibited enzyme. The reduction potential of cytochrome a is only moderately (less than -30 mV/pH unit) dependent upon pH, which implies that its reduction is linked to the uptake, on the average, of only about 0.5 protons at pH 7.0, and significantly less at the higher pH values relevant to the mitochondrial matrix. The thermodynamic properties of cytochrome a3 were found to be different in the two enzyme batches studied: in one batch, the cytochrome a3 reduction potential decreased steeply (about -56 mV/pH unit) with increasing pH, indicating stoichiometric (1 H+/e-) coupling of protonation to reduction. In the other batch, the cytochrome a3 potential was insensitive to pH below pH 7.5 and decreased at higher pH values in a manner suggesting coupling to an ionizable group with pKa near 7.8. The temperature dependence of the cytochrome a3 reduction potential indicates that its standard entropy of reduction is more positive than that of myoglobin, another high-spin metalloprotein heme, and significantly more positive than that of cytochrome a.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.     

pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes.

1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox characterization of Geobacter sulfurreducens cytochrome c7: physiological relevance of the conserved residue F15 probed by site-specific mutagenesis

The complete genome sequence of the delta-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes. Cytochrome c(7), isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with M(r) 9.6 kDa. This protein is involved in metal ion-reducing pathways and shares 56% sequence identity with a triheme cytochrome isolated from the closely related delta-proteobacterium Desulfuromonas acetoxidans (Dac(7)). In this work, two-dimensional NMR was used to monitor the heme core and the general folding in solution of the G. sulfurreducens triheme cytochrome c(7) (PpcA). NMR signals obtained for the three hemes of PpcA at different stages of oxidation were cross-assigned to the crystal structure [Pokkuluri, P. R., Londer, Y. Y., Duke, N. E. C., Long, W. C., and Schiffer, M. (2004) Biochemistry 43, 849-859] using the complete network of chemical exchange connectivities, and the order in which each heme becomes oxidized was determined at pH 6.0 and 8.2. Redox titrations followed by visible spectroscopy were also performed in order to monitor the macroscopic redox behavior of PpcA. The results obtained showed that PpcA and Dac(7) have different redox properties: (i) the order in which each heme becomes oxidized is different; (ii) the reduction potentials of the heme groups and the global redox behavior of PpcA are pH dependent (redox-Bohr effect) in the physiological pH range, which is not observed with Dac(7). The differences observed in the redox behavior of PpcA and Dac(7) may account for the different functions of these proteins and constitute an excellent example of how homologous proteins can perform different physiological functions. The redox titrations followed by visible spectroscopy of PpcA and two mutants of the conserved residue F15 (PpcAF15Y and PpcAF15W) lead to the conclusion that F15 modulates the redox behavior of PpcA, thus having an important physiological role.     

Site-specific mutagenesis of annexin V: role of residues from Arg-200 to Lys-207 in phospholipid binding.

Annexin V (placental anticoagulant protein I) binds tightly to anionic phospholipid vesicles in the presence of calcium. Four mutant proteins were expressed in Escherichia coli in which Ala replaced one of the following residues in the third repeat of annexin V: Arg-200, His-204, Arg-206, or Lys-207. In a competitive fluorescence quenching assay, the wild-type recombinant protein had the same affinity for phosphatidylserine-containing vesicles as the placentally derived protein. The affinity of the four mutant proteins for phosphatidylserine-containing vesicles was unchanged relative to wild-type protein. We conclude that His-204 and adjacent basic residues, including the highly conserved Arg-200 residue, are not required for high-affinity phospholipid binding.     

Similarities in structure between holocytochrome b5 and apocytochrome b5: NMR studies of the histidine residues

The properties of the six histidine residues of apocytochrome b5 have been investigated by using one- and two-dimensional proton NMR spectroscopy in order to probe the structure remaining after heme removal. Spectral assignments were arrived at by analyzing proton NOE connectivities, comparing them to those observed in the holoprotein, and inspecting the X-ray structure of the latter species. Each histidine residue was studied for its pKa value, interaction with the relaxation agent copper nitrilotriacetic acid, and reactivity toward bromoacetic acid. The four histidines which are not coordinated to the iron atom in the holoprotein (His-15, -26, -27, and -80) display in the major conformer of the apoprotein the same characteristic properties as in the holoprotein. Three of them are involved in specific interactions with the rest of the structure: His-15 and His-80 participate in hydrogen bonds, and His-27 is influenced by the nearby C-terminal segment. His-26 is the most exposed to the solvent. His-63 and His-39, which are located in the heme binding site, have distinct pKa values; they are affected differently by the copper agent and exhibit comparable reactivity toward bromoacetic acid, albeit milder than that of His-26. The results show that the heme binding residues are clearly distinguishable by their physicochemical properties and that several elements of native holoprotein structure are in place in the apoprotein. It is proposed that the structural influence of the heme is localized and that the amino- and carboxy-terminal segments form a structural unit providing stability to the apoprotein and supporting a fluctuating, partially folded binding site.