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M S Caffrey

Publications and source records attributed to M S Caffrey.

At least 19 recordsLinked to original sources
PubMed1997

The substitution of proline 35 by alanine in Rhodobacter capsulatus cytochrome c2 affects the overall protein stability but not the alkaline transition.

It was shown by Koshy et al. [1990, Proc. Natl Acad. Sci USA, 87, 8697-8701; 1994, Biochem. J., 299, 347-350] that the substitution of proline 30 by alanine (P30A) of Drosophila melanogaster and rat cytochromes c exhibited decreased stabilities in both the heme iron-methionine sulfur (Fe-S) bond and overall protein conformation. Now we have found that the stability properties of the equivalent mutant of Rhodobacter capsulatus cytochrome c2 (P35A) are somewhat different. Based on optical and NMR spectroscopies, the Rb.capsulatus P35A alkaline transition (pKalk) was found to be unchanged with respect to the wild type, suggesting that the mutation in Rb.capsulatus cytochrome c2 has little effect on the stability of the Fe-S bond. However, Rb.capsulatus conformational stability was found to be decreased by 1.6 kcal/mol in the oxidized state. The difference in the stability properties of the equivalent proline to alanine substitutions in various species underscores the importance of studying mutations in more than one species before drawing generalizations about the role of conserved residues in protein structure and function.

Cytochrome c Group↗
PubMed1995

Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation.

The stability properties of oxidized wild-type (wt) and site-directed mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) ferredoxins from Anabaena 7120 have been characterized by guanidine hydrochloride (Gdn-HCl) denaturation. For Vfd it was found that mutants E95K, E94Q, F65Y, F65W, and T48A are quite similar to wt in stability. E94K is somewhat less stable, whereas E94D, F65A, F65I, R42A, and R42H are substantially less stable than wt. R42H is a substitution found in all Hfds, and NMR comparison of the Anabaena 7120 Vfd and Hfd showed the latter to be much less stable on the basis of hydrogen exchange rates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemistry 33:3287-3295); we also find this to be true with respect to Gdn-HCl denaturation. Strikingly, the Hfd mutant H42R is more stable than the wt Hfd by precisely the amount of stability lost in Vfd upon mutating R42 to H (2.0 kcal/mol). On the basis of comparison of the X-ray crystal structures of wt Anabaena Vfd and Hfd, the decreased stabilities of F65A and F65I can be ascribed to increased solvent exposure of interior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the decreased stabilities may result from disruption of a hydrogen bond between the E94 and S47 side chains. The instability of the R42 mutants is also most probably due to decreased hydrogen bonding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)

Anabaena↗
PubMed1994

Assignment of NMR spectra of proteins using triple-resonance two-dimensional experiments.

Two-dimensional versions of HNCA and HNCO experiments are described, which provide essentially the same information as the 3D sequence. A multiple-quantum coherence involving either 15N and 13C alpha or 15N and 13CO is created. One of the two frequencies is given by the middle point between the two cross peaks (zero- and double-quantum) and the other by their separation. Quadrature detection can be performed on either nucleus, modifying only the appearance of the 2D spectrum, but not the information content. These experiments, named MQ-HNCA and MQ-HNCO, are illustrated on a (15N, 13C) doubly labelled cytochrome c2 from Rhodobacter capsulatus (116 amino acids).

Carbon Isotopes↗
PubMed1994

Strategies for the study of cytochrome c structure and function by site-directed mutagenesis.

The class I cytochromes c have been extensively studied by biochemical and biophysical methods; however, many questions remain concerning the roles of specific amino acids in electron transfer and stability properties. The method of site-directed mutagenesis, which substitutes specific amino acid residues by genetic methods, is ideal for addressing these questions of cytochrome c structure and function. Practical considerations of mutational effects on protein processing and stability will be addressed. The criteria for the selection of mutation sites will be discussed. Examples of site-directed mutagenesis studies, which were designed to elucidate the factors controlling biological electron transfer, protein processing, and protein stability, are given.

Amino Acid Sequence↗
PubMed1993

The interaction between cytochrome c2 and the cytochrome bc1 complex in the photosynthetic purple bacteria Rhodobacter capsulatus and Rhodopseudomonas viridis.

The rates of electron transfer from a ubiquinol analogue to cytochrome c2 catalyzed by the cytochrome bc1 complexes of Rhodobacter capsulatus and Rhodopseudomonas viridis were measured as a function of ionic strength. The effects of ionic strength on the kinetic parameters for the reactions are consistent with a role for electrostatic complex formation between cytochrome c2 and the cytochrome bc1 complex in the electron-transfer pathways in both photosynthetic purple non-sulfur bacteria. Additional support for a docking model in which positively charged lysines on cytochrome c2 interact with negatively charged groups on the Rb. capsulatus cytochrome bc1 complex was obtained from kinetic experiments using Rb. capsulatus cytochrome c2 and equine cytochrome c in which specific lysine residues were altered by site-directed mutagenesis and chemical modification, respectively. Equine cytochrome c, which is a poor electron donor to the reaction center of Rps. viridis, is an effective electron acceptor for the Rps. viridis cytochrome bc1 complex. Chemical modification of lysine residues on Rps. viridis cytochrome c2 has a substantially greater effect on the reduction of the Rps. viridis reaction center by ferrocytochrome c2 than on the oxidation of the Rps. viridis cytochrome bc1 complex by ferricytochrome c2. These data suggest that the docking site for Rps. viridis cytochrome c2 on the Rps. viridis reaction center tetraheme subunit differs in structure from the docking site for the cytochrome on the Rps. viridis cytochrome bc1 complex to a significant extent. In this respect, Rps. viridis differs from photosynthetic purple non-sulfur bacteria in which the reaction center does not contain a tetraheme subunit, where the binding sites for cytochrome c2 on the reaction center and the cytochrome bc1 complex appear to be quite similar.

Chromatography, Gel↗
PubMed1993

Purification and properties of an unusual membrane-derived cytochrome b-561 from the purple phototrophic bacterium Rhodobacter capsulatus, which is structurally related to the bacteriochlorophyll-binding protein, LHII beta.

An abundant cytochrome b-561 was solubilized from Rhodobacter capsulatus membranes by successive treatments with perchlorate and butanol/water. Neither procedure was effective alone although they could be combined into a single step. Once solubilized, cytochrome b-561 was purified by standard chromatographic procedures used for water-soluble proteins without addition of butanol or detergents. Cytochrome b-561 appears to be highly acidic, it has a size greater than about 1000 kDa as isolated, and the subunit size measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is less than 8 kDa. The redox potential measured by cyclic voltammetry is -65 mV at pH 7. The N-terminal amino acid sequence is identical to that of the Rb. capsulatus LHII beta light-harvesting bacteriochlorophyll binding protein subunit which has only 48 amino acid residues, and the mass, determined by mass spectroscopy, is identical to that of LHII beta. There is but one heme per two to three peptide chains of 5 kDa, which suggests that the two extraplanar ligands to the heme are on separate subunits. There is strong exciton splitting in the circular dichroism spectrum in the Soret region indicative of heme-heme interaction. The helix content based on far-uv CD is 41%. Together, these properties of cytochrome b-561 are very similar to those of isolated LHII alpha beta bacteriochlorophyll-protein complexes.

Amino Acid Sequence↗
PubMed1993

Role of the highly conserved tryptophan of cytochrome c in stability.

To test the importance of the highly conserved tryptophan to the structure and function of cytochrome c, we have prepared the mutant W67Y (tryptophan 67 substituted by tyrosine) of Rhodobacter capsulatus cytochrome c2. The mutation appears to have little effect on the redox potential or in vitro electron transfer properties. The presence of the W67Y near-infrared absorbance at 702 nm establishes that methionine is ligated to the heme group but the 6-nm red-shift in the absorbance maxima suggests that the environment of the heme iron-methionine sulfur bond is perturbed. Moreover, the pK for W67Y alkaline transition is decreased 0.9 pH units with respect to the wild-type, indicating that the heme iron-methionine sulfur bond of the mutant is destabilized approximately 1 kcal/mol. Based on guanidine-HCl denaturation monitored with the 220-nm circular dichroism signal, the W67Y conformational stability is decreased by 40% (2.5 kcal/mol) and 60% (3.0 kcal/mol) in the oxidized and reduced states, respectively. Collectively, these data suggest that tryptophan 67 of Rb. capsulatus cytochrome c2, or its equivalent in other species, is important to stability but is not critical to the time-averaged structure, redox potential, or function.

Bacterial Proteins↗
PubMed1992

Study of the cytochrome c2-reaction center interaction by site-directed mutagenesis.

Photooxidation of Rhodobacter capsulatus cytochrome c2 and four site-directed mutants by detergent solubilized Rhodobacter sphaeroides reaction centers was studied as a function of ionic strength at pH 8.0. Mutants of cytochrome c2 included K12D (lysine 12 substituted by aspartate), K14E (lysine 14 substituted by glutamate), K32E (lysine 32 substituted by glutamate), and K14E/K32E (lysines 14 and 32 substituted by glutamates). With respect to the wild-type, the mutants exhibited decreased second-order rate constants, indicating perturbation of their electrostatic interaction with the reaction center. In the transient complex, the interaction domain charges of the reaction center and wild-type cytochrome c2 were estimated to be -4.8 and +4.8, respectively. In contrast, the interaction domain charges of mutants K12D, K14E, K32E, and K14E/K32E were estimated to be +2.8, +3.7, +3.6 and +1.3, respectively. At infinite ionic strength, the second-order rate constant of the wild-type cytochrome c2 photooxidation (k infinity) was estimated to be 8.7 x 10(6) M-1 s-1. In the case of K32E, k infinity was not changed significantly (8.2 x 10(6) m-1 s-1), suggesting that the electrostatic perturbation of this mutant was largely overcome at high ionic strength. In contrast, the k infinity for K12D, K14E, and K14E/K32E were estimated to be decreased 2-7-fold. Consequently, mutations to R. capsulatus lysines 12 and 14 appear to perturb the distance and/or orientation of the cytochrome c2 relative to the reaction center in the reactive complex, as well as alter electrostatic interactions. Based upon the kinetic results presented here, the cytochrome c2-reaction center transient complex has been modeled.

Amino Acid Sequence↗
PubMed1992

Mutations Pro----Ala-35 and Tyr----Phe-75 of Rhodobacter capsulatus ferrocytochrome c2 affect protein backbone dynamics: measurements of individual amide proton exchange rate constants by 1H-15N HMQC spectroscopy.

Comparisons of hydrogen-deuterium solvent exchange rate constants for the NH protons of wild-type Pro----Ala-35 (P35A) and Tyr----Phe-75 (Y75F) Rhodobacter capsulatus ferrocytochromes c2 were made by 1H-15N heteronuclear multiple-quantum correlation spectroscopy. Exchange rate constants increased for the NH protons of residues 45-46, 54, 57-58, 60-61, 82-87, 98, and 100 with Y75F and 16-18, 20, 34, 37, 43, 45-46, and 58 with P35A. The increases in exchange rate constants are consistent with changes in unfolding equilibria and protein dynamics. In Y75F the exchange rate constants of the observable NH protons of the helix spanning Pro-79-Asp-89, namely Phe-82-Leu-87, increase to a similar degree, suggesting that this helix is a single cooperative unfolding unit compatible with the local unfolding model. As the oxidation-reduction potential of Y75F is 59 mV lower than wild-type cytochrome c2 (367 mV), the dynamic changes in this mutant, compared to wild-type, are proposed to be important determinants of the oxidation-reduction potential. Several differences between wild-type and Y75F are in common with P35A, a mutation which does not affect the oxidation-reduction potential, implying that not all observed dynamic changes are functionally important.

Amides↗
PubMed1991

Lysines in the amino-terminal alpha-helix are important to the stability of Rhodobacter capsulatus cytochrome c2.

The protein stabilities of wild type and four site-directed mutants of Rhodobacter capsulatus cytochrome c2 have been characterized. The integrity of the cytochrome c2 iron-sulfur environment was ascertained by titration of the 696-nm absorbance band with alkali, and the conformational stability was determined by titration of the 220-nm circular dichroism signal with Gdn-HCl. Analysis of the alkaline transition pK value of K12D (lysine-12 substituted by aspartate) indicated that the K12D iron-sulfur environment was destabilized by 0.6 kcal/mol relative to the wild-type cytochrome c2 at low ionic strength. In contrast, the alkaline transition pK values of K14E (lysine-14 substituted by glutamate), K32E (lysine-32 substituted by glutamate), and K14E/K32E (lysines-14 and -32 substituted by glutamates) were indistinguishable from the wild type, indicating that these substitutions have no effect on the stability of the iron-sulfur environment. Gdn-HCl denaturation of K12D and K14E indicated that both these mutations decreased conformational stability by 1.3 kcal/mol. In contrast, mutant K32E exhibited a small stabilizing effect of 0.2 kcal/mol. Gdn-HCl denaturation of K14E/K32E indicated that this mutation decreased conformational stability by 1.3 kcal/mol, which is consistent with the additive effects of the single charge mutations at positions 14 and 32. The conformational instability of mutants possessing negative charges at position 12 or 14 is best explained by their positioning at the carboxy-terminal region of the amino-terminal alpha-helix of R. capsulatus cytochrome c2. Accordingly, introduction of negatively charged groups into this region appears to destabilize cytochrome c2 through energetically unfavorable interactions with the dipole of the amino-terminal helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2.5 A resolution.

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.

Amino Acid Sequence↗
PubMed1991

Biological electron transfer: progress and future directions.

The rich diversity among bacterial cytochromes has played a key role in the development of our understanding of biological electron transfer. Although studies to date have allowed the elucidation of the contributions of driving force, electrostatics interactions and surface topology to electron transfer kinetics in collision-dependent reactions, much remains to be learned. Little is known about intramolecular and intracomplex electron transfer. Several factors controlling intramolecular and intracomplex electron transfer can be defined. These include driving force, the distance between redox centers, the relative orientation of prosthetic groups, the nature of the intervening media and the molecular dynamics within the electron transfer complex. However, at the present time, we have only a limited understanding of the contribution of these factors to electron transfer kinetics in biologically relevant systems. Nevertheless, a wide range of techniques are now available which should soon provide the information necessary to describe in molecular terms the mechanism of intramolecular and intracomplex electron transfer. Principal among these new approaches are site-directed mutagenesis and NMR spectroscopy.

Animals↗
PubMed1991

Redox potentials of flavocytochromes c from the phototrophic bacteria, Chromatium vinosum and Chlorobium thiosulfatophilum.

The redox potentials of flavocytochromes c (FC) from Chromatium vinosum and Chlorobium thiosulfatophilum have been studied as a function of pH. Chlorobium FC has a single heme which has a redox potential of +98 mV at pH 7 (N = 1) that is independent of pH between 6 and 8. The average two-electron redox potential of the flavin extrapolated to pH 7 is +28 mV and decreases 35 mV/pH between pH 6 and 7. The anionic form of the flavin semiquinone is stabilized above pH 6. The redox potential of Chromatium FC is markedly lower than for Chlorobium. The two hemes in Chromatium FC appear to have a redox potential of 15 mV at pH 7 (N = 1), although they reside in very different structural environments. The hemes of Chromatium FC have a pH-dependent redox potential, which can be fit in the simplest case by a single ionization with pK = 7.05. The flavin in Chromatium FC has an average two-electron redox potential of -26 mV at pH 7 and decreases 30 mV/pH between pH 6 and 8. As with Chlorobium, the anionic form of the flavin semiquinone of Chromatium FC is stabilized above pH 6. The unusually high redox potential of the flavin, a stabilized anion radical, and sulfite binding to the flavin in both Chlorobium and Chromatium FCs are characteristics shared by the flavoprotein oxidases. By analogy with glycolate oxidase and lactate dehydrogenase for which there are three-dimensional structures, the properties of the FCs are likely to be due to a positively charged amino acid side chain in the vicinity of the N1 nitrogen of the flavin.

Bacteria↗
PubMed1991

Expression of the gene encoding cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris in the purple photosynthetic bacterium Rhodobacter sphaeroides.

The gene encoding cytochrome c3 (cyc-gene) from Desulfovibrio vulgaris (Hildenborough) was cloned by G. Voordouw and S. Brenner (1986, Eur. J. Biochem. 159, 347-351). The gene was expressed in Escherichia coli but only the apoprotein was observed (W. Pollock, P. Chemerika, M. Forrest, J. Beatty, and G. Voordouw, 1989, J. Gen. Microbiol. 135, 2319-2328). In this study, the cyc-gene was cloned into the broad host range vector pRK404 and then introduced into the purple photosynthetic bacterium Rhodobacter sphaeroides. Cells grown anaerobically produced a significant amount of recombinant cytochrome c3. The purified protein contains four hemes and the N-terminal protein sequence is identical to the published sequence of the native cytochrome c3. Thus, R. sphaeroides was able to produce the mature cytochrome c3 by combining the four steps of protein synthesis, exporting the protein across the membrane, cleaving the signal peptide, and inserting four hemes. It appears that the D. vulgaris promoter is not very efficiently used by R. sphaeroides. However, replacement of the promoter with a R. sphaeroides promoter should result in cytochrome c3 overproduction.

Cloning, Molecular↗
PubMed1991

Importance of a conserved hydrogen-bonding network in cytochromes c to their redox potentials and stabilities.

To understand the determinants of redox potential and protein stability in c-type cytochromes, we have characterized two mutations to a highly conserved tyrosine group, tyrosine-75, of Rhodobacter capsulatus cytochrome c2. Mutant Y75F was designed to test the importance of the tyrosine hydroxyl group to the typically high redox potentials of the cytochromes c2 while maintaining a hydrophobic core. Mutant Y75C was designed to test the importance of a large hydrophobic group to redox potential by replacing an aromatic group with a small nonpolar group. Both mutants exhibit spectral and redox properties indicating that their heme environments have been perturbed. The kinetics of reduction by lumiflavin semiquinone and photooxidation by Rhodobacter sphaeroides photosynthetic reaction centers have been used to demonstrate that both mutants are structurally analogous to the wild-type protein at the active site of electron transfer. Different degrees of relative stability of the mutants toward a denaturant have been observed with the order being Y75C less than wt less than Y75F in the oxidized state and Y75C less than Y75F less than wt in the reduced state. These results are discussed in light of the recent structure determination of the R. capsulatus wild-type ferrocytochrome c2 to suggest that R. capsulatus tyrosine-75, or its equivalent in other species, is part of a conserved hydrogen-bonding network which plays an important role in maintaining high redox potentials and protein stability of cytochromes c in general.

Circular Dichroism↗
PubMed1991

A spectroscopic analysis of the Pro35----Ala mutant of Rhodobacter capsulatus cytochrome c2. The strictly conserved Pro35 is not structurally essential.

Visible, near-ultraviolet circular dichroic, near-infrared and nuclear magnetic resonance spectroscopies show that the secondary and tertiary structures of the mutant Pro35----Ala Rhodobacter capsulatus ferrocytochrome c2 are similar to the wild-type protein. The near-infrared spectrum shows that the methionine-S--Fe-heme bond is intact; however, a small red shift in the heme M transition of the near-ultraviolet circular dichroic spectrum of the mutant indicates that the heme environment may differ slightly between the two proteins. This difference may be a consequence of changes in the ligand and hydrogen bonds of His17 [Gooley, P. R. & MacKenzie, N. E. (1990) FEBS Lett. 260, 225-228]. 1H and 15N chemical shift differences suggest that the microenvironment of pyrrole rings III and IV of the heme prosthetic group differs between the two proteins. As the rings of the Phe51 and Tyr53 flip faster in the mutant protein than the wild type, these chemical shift differences may reflect changes in the time-average ring-current effects and not structural alterations.

Circular Dichroism↗
PubMed1991

The effects of surface charges on the redox potential of cytochrome c2 from the purple phototrophic bacterium Rhodobacter capsulatus.

Four site-directed mutants of Rhodobacter capsulatus cytochrome c2, which substitute lysines at three positions with aspartate or glutamate, have been prepared. Mutations included the single charge substitutions K12D, K14E, and K32E and a double charge substitution K14E/K32E. Characterization of the ionic strength dependence of the wild-type and mutant redox potentials in the "nonbinding" buffer Tris-cacodylate suggests that (i) at zero ionic strength introduction of negatively charged groups stabilizes the oxidized state by 11-14 mV per charge and (ii) at high ionic strengths where the charged groups are masked, the effects of single charge substitutions are overcome; however, the redox potential of the double charge substitution is still affected. These results indicate that at physiological ionic strengths charge distribution only affects redox potential when the heme environment has been perturbed by a structural perturbation and that the determinants of redox potential in c-type cytochromes is primarily due to the local heme environment.

Aspartic Acid↗