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Heme c is characterized by its covalent attachment to a polypeptide. The attachment is typically to a CXXCH motif in which the two Cys form thioether bonds with the heme, “X” can be any amino acid other than Cys, and the His serves as a heme axial ligand. Some cytochromes c, however, contain heme attachment motifs with three or four intervening residues in a CX3CH or CX4CH motif. Here, the impacts of these variations in the heme attachment motif on heme ruffling and electronic structure are investigated by spectroscopically characterizing CX3CH and CX4CH variants of Hydrogenobacter thermophilus cytochrome c552. In addition, a novel CXCH variant is studied. 1H and 13C NMR, EPR, and resonance Raman spectra of the protein variants are analyzed to deduce the extent of ruffling using previously reported relationships between these spectral data and heme ruffling. In addition, the reduction potentials of these protein variants are measured using protein film voltammetry. The CXCH and CX4CH variants are found to have enhanced heme ruffling and lower reduction potentials. Implications of these results for the use of these noncanonical motifs in nature, and for the engineering of novel heme peptide structures, are discussed. [ABSTRACT FROM AUTHOR]

Heme axial methionine ligands in ferricytochromes c552 from Hydrogenobacter thermophilus (HT) and Nitrosomonas europaea, both members of the cyt c6 family, display fluxional behavior. The ligand motion, proposed to be inversion at sulfur, results in an unusually small range of hyperfine shifts for heme substituents in these proteins. Herein, heme axial Met fluxion is induced in a structurally homologous cytochrome c6 from Pseudomonas aeruginosa (PA) by substituting heme pocket residue Asn64 with Gln. The mutant, PA-N64Q, displays a highly compressed range of heme substituent hyperfine shifts, temperature-dependent heme methyl resonance line broadening, low rhombic magnetic anisotropy, and a magnetic axes orientation consistent with Met orientational averaging. Analysis of NMR properties of PA-N64Q demonstrates that the heme pocket of the mutant resembles that of HT. This result confirms the importance of peripheral interactions and, in particular, residue 64 in determining axial Met orientation and heme electronic structure in proteins in the cyt c6 family. [ABSTRACT FROM AUTHOR]

Proteins in the cytochrome c (cyt c) family with His-Met heme axial ligation display diverse heme electronic structures as revealed by the NMR spectra of their oxidized (paramagnetic) forms. These variations in electronic structure are thought to result primarily from differences in heme axial Met orientation among cyt c species. The factors determining Met orientation in cyts c, however, remain poorly understood. An additional layer of complexity was revealed with the recent finding that the axial Met in Hydrogenobacter thermophilus cytochrome C552 (Ht cyt c552) is fluxional, sampling two conformations rapidly on the NMR time scale, resulting in an unusual compressed range of heme substituent hyperfine shifts [Zhong, L., Wen, X., Rabinowitz, T. M., Russell, B. S., Karan, E. F., and Bren, K. L. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 8637-8642]. In this work, the ¹H NMR hyperfine shift pattern of Ht cyt c552 is drastically altered by making the conservative heme pocket mutation Gln64Asn. The mutant (Ht Q64N) displays a pattern of heme hyperfine shifts with a remarkable resemblance to that of structurally homologous Pseudomonas aeruginosa cyt C551, which has Asn at position 64 and a single heme axial Met conformation. NMR analysis reveals that Asn64 in Ht Q64N is positioned to interact with the axial Met61, whereas the G1n64 in wild-type Ht cyt c552 is not. It also is found that the heme axial Met is not flux ional in Ht Q64N and has an orientation similar to that in P. aeruginosa cyt C551. These results indicate that peripheral interactions with the axial Met play an important role in determining axial Met orientation and heme electronic structure in cyts c. [ABSTRACT FROM AUTHOR]

NMR spectroscopy has become a vital tool for studies of protein conformational changes and dynamics. Oxidized Fe(lll)cytochromes c are a particularly attractive target for NMR analysis because their paramagnetism (S = ½) leads to high 1H chemical shift dispersion, even for unfolded or otherwise disordered states. In addition, analysis of shifts induced by the hypertine interaction reveals details of the structure of the heme and its ligands for native and nonnative protein conformational states. The use of NMR spectroscopy to investigate the folding and dynamics of paramagnetic cytochromes c is reviewed here. Studies of nonnative conformations formed by denaturation and by anomalous in vivo maturation (heme attachment) are facilitated by the paramagnetic, low-spin nature of native and nonnative forms of cytochromes c. Investigation of the dynamics of folded cytochromes c also are aided by their paramagnetism. As an example of this analysis, the expression in Escherichia coli of cytochrome c552 from Nitrosornonas europaea is reported here, along with analysis of its unusual heme hyperfine shifts. The results are suggestive of heme axial methionine fluxion in N. europaea ferricytochrome c552. The application of NMR spectroscopy to investigate paramagnetic cytochrome c folding and dynamics has advanced our understanding of the structure and dynamics of both native and nonnative states of heme proteins. [ABSTRACT FROM AUTHOR]

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c551 dynamics based on 15N R1, 15N R2, and {1H}-15N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (2>=0.924±0.005) and displays little variation in picosecond–nanosecond time scale dynamics over the structure. The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content. Also reported are protection factors calculated from hydrogen-exchange rates. These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions. These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (2>=0.80±0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3. This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0401-z. On that page (frame on the left side), a link takes you directly to the supplementary material. [ABSTRACT FROM AUTHOR]

Cytochromes c are characterized by the presence of a protoporphyrin IX group covalently attached to the polypeptide via one or two thioether bonds to Cys side chains. The heme attachment process, known as cytochrome c maturation, occurs posttranslationally in the periplasm (for bacterial cytochromes c) or in the mitochondrial intermembrane space (for eukaryotic cytochromes c) through a pathway dependent on the organism. It is demonstrated in this work that a mitochondrial cytochrome c expressed in Escherichia coli that undergoes maturation under control of the E. coli cytochrome c maturation factors achieves a native-like structure and stability. The recombinant protein is characterized spectroscopically (by circular dichroism (CD), absorption, and nuclear magnetic resonance (NMR) spectroscopy) and it is verified that the heme and its environment are indistinguishable from authentic horse cytochrome c. Mass spectrometry reveals that the recombinant protein is not acetylated at the N terminus, however, no significant effect on protein structure or stability is detected as a result. [Copyright &y& Elsevier]

Reports on structurally engineered cytochromes with novel ligand-binding sites. Employment of semisynthesis to manipulate the structure of the heme pocket of horse heart cytochrome; Occurrence of the cyt c refolding in the absence of a position-80 ligand; Engineering of a cavity in the position-80 region of cyt c.

The heme in cytochromes c undergoes a conserved out-of-plane distortion known as ruffling. For cytochromes c from the bacteria Hydrogenobacter thermophilus and Pseudomonas aeruginosa, NMR and EPR spectra have been shown to be sensitive to the extent of heme ruffling and to provide insights into the effect of ruffling on the electronic structure. Through the use of mutants of each of these cytochromes that differ in the amount of heme ruffling, NMR characterization of the low-spin (S = 1/2) ferric proteins has confirmed and refined the developing understanding of how ruffling influences the spin distribution on heme. The chemical shifts of the core heme carbons were obtained through site-specific labeling of the heme via biosynthetic incorporation of 13C-labeled 5-aminolevulinic acid derivatives. Analysis of the contact shifts of these core heme carbons allowed Fermi contact spin densities to be estimated and changes upon ruffling to be evaluated. The results allow a deconvolution of the contributions to heme hyperfine shifts and a test of the influence of heme ruffling on the electronic structure and hyperfine shifts. The data indicate that as heme ruffling increases, the spin densities on the β-pyrrole carbons decrease while the spin densities on the α-pyrrole carbons and meso carbons increase. Furthermore, increased ruffling is associated with stronger bonding to the heme axial His ligand. [ABSTRACT FROM AUTHOR]

Out-of-plane (OOP) deformations of the heme cofactor are found in numerous heme-containing proteins and the type of deformation tends to be conserved within functionally related classes of heme proteins. We demonstrate correlations between the heme ruffling OOP deformation and the 13C and ¹H nuclear magnetic resonance (NMR) hyperfine shifts of heme aided by density functional theory (DFT) calculations. The degree of ruffling in the heme cofactor of Hydrogenobacter thermophilus cytochrome c552 has been modified by a single amino acid mutation in the second coordination sphere of the cofactor. The 13C and ¹H resonances of the cofactor have been assigned using one- and two-dimensional NMR spectroscopy aided by selective 13C-enrichment of the heme. DFT has been used to predict the NMR hyperfine shifts and electron paramagnetic resonance (EPR) g-tensor at several points along the ruffling deformation coordinate. The DFT-predicted NMR and EPR parameters agree with the experimental observations, confirming that an accurate theoretical model of the electronic structure and its response to ruffling has been established. As the degree of ruffling increases, the heme methyl ¹H resonances move upfield while the heme methyl and meso 13C resonances move downfield. These changes are a consequence of altered overlap of the Fe 3d and porphyrin π orbitals, which destabilizes all three occupied Fe 3d-based molecular orbitals and decreases the positive and negative spin density on the β-pyrrole and meso carbons, respectively. Consequently, the heme ruffling deformation decreases the electronic coupling of the cofactor with external redox partners and lowers the reduction potential of heme. [ABSTRACT FROM AUTHOR]

Electron paramagnetic resonance (EPR) spectra of variants of Hydrogenobacter thermophilus cytochrome c552 (Ht c-552) and Pseudomonas aeruginosa cytochrome c551 (Pa c-551) are analyzed to determine the effect of heme ruffling on ligand-field parameters. Mutations introduced at positions 13 and 22 in Ht c-552 were previously demonstrated to influence hydrogen bonding in the proximal heme pocket and to tune reduction potential (Em) over a range of 80 mV [Michel, L. V.; Ye, T.; Bowman, S. E. J.; Levin, B. D.; Hahn, M. A.; Russell, B. S.; Elliott, S. J.; Bren, K. L. Biochemistry2007, 46, 11753-11760]. These mutations are shown here to also increase heme ruffling as Em decreases. The primary effect on electronic structure of increasing heme ruffling is found to be a decrease in the axial ligand-field term Δ/λ, which is proposed to arise from an increase in the energy of the dxy orbital. Mutations at position 7, previously demonstrated to influence heme ruffling in Pa c-551 and Ht c-552, are utilized to test this correlation between molecular and electronic structure. In conclusion, the structure of the proximal heme pocket of cytochromes c is shown to play a role in determining heme conformation and electronic structure. [ABSTRACT FROM AUTHOR]

The direct electrochemical analysis of adsorbed redox active proteins has proven to be a powerful technique in biophysical chemistry, frequently making use of the electrode material pyrolytic “edge-plane” graphite. However, many heme-bearing proteins such as cytochromes chave been also examined systematically at alkanethiol-modified gold surfaces, and previously we reported the characterization of the redox properties of a series of bacterial cytochromes cin a side-by-side comparison of carbon and gold electrode materials. In our prior findings, we reported an unanticipated, low potential (Em∼ −100 mV vs SHE) redox couple that could be analogously observed when a variety of monoheme cytochromes care adsorbed onto carbon-based electrodes. Here we demonstrate that our prior phenomological data can be understood quantitatively in the loss of the methionine ligand of the heme iron, using the cytochrome cfrom Hydrogenbacter thermophilumas a model system. Through the comparison of wild-type protein with M61H and M61A mutants, in direct electrochemical analyses conducted as a function of temperature and exogenous ligand concentration, we are able to show that Met-ligated cytochromes chave a propensity to lose their Met ligand at graphite surfaces, and that energetics of this process (6.3 ± 0.2 kJ/mol) is similar to the energies associated with “foldons” of known protein folding pathways. [ABSTRACT FROM AUTHOR]

Unfolding thermodynamics of a thermophilic cytochrome C552 from Hydrogenobacter thermophilus (Ht cyt c552) and its mesophilic homologue from Pseudomonas aeruginosa (Pa cyt c551) as well as two heme pocket point mutants (Ht-Q64N and Pa-N64Q) are characterized by determination of protein stability curves (plots of unfolding free energy, ΔG, vs T). These proteins show revealing differences in heme pocket hydrogen bonding and mobility. It previously has been shown that Asn64 in Pa cyt c551 and in Ht-Q64N interacts with the heme axial Met to fix it in a single conformation [Wen, X., and Bren, K. L. (2005) Biochemistry 44, 5225-5233]. In Ht cyt c552 and Pa-N64Q, Gln64 does not interact with the axial Met; in these variants the axial Met samples more than one conformation [Wen, X., and Bren, K. L. (2005) Inorg. Chem. 44, 8587-8593]. Here it is demonstrated that, relative to wild type, Pa-N64Q displays enhanced stability with an increase in unfolding free energy (ΔΔG) of 7.1 kJ/mol and an increase in denaturation temperature (ΔTm) of 8 °C. Correspondingly, Ht-Q64N is less stable than Ht cyt c552, with a ΔΔG of -10 kJ/mol and a ΔTm of -10 °C. Analysis of unfolding thermodynamics indicates that the net changes in stability resulting from the position 64 mutations are primarily attributable to entropic factors. For Pa-N64Q (Ht-Q64N) it is proposed that the favorable release (unfavorable burial) of residue 64 is the dominant factor impacting stability. The mobility of the axial Met also is proposed to contribute. These results provide a specific illustration of how amino acid side chain mobility and burial or release contribute to protein stability. [ABSTRACT FROM AUTHOR]

We have used protein film voltammetry (PFV) to determine the midpoint potentials of the Pseudomonas aeruginosa, Hydrogenobacter thermophilus, and Nitrosomonas europaaa wild-type monoheme cytochromes c (cyts c; PA, HT, and NE, respectively), as well as PA N64Q, HT Q64N, and NE V65Δ mutants, as a function of pH, and buffer conditions. Recent studies have suggested that the identity of the 64 position of the heme-binding loop (either Asn or Gln) strongly influences the conformation of the Met ligand that binds the heme iron. The PFV studies reveal that HT and NE possess significantly lower potentials (wild-type cyts c having Em values of +227 and +250 mV vs SHE) than PA (+290 mV) in 50 mM phosphate buffer, pH 7 at 3 °C. The HT Q64N mutant rises in potential compared to wild-type, and the PA N64Q mutant has a lower potential, indicating relationships between Met ligand fluxion, hydrogen bonding to the Met ligand, and redox chemistry. Surprisingly, NE V65Δ, possessing a heme binding loop nearly identical to that of the PA protein, displayed an Em of + 232 mV, even lower than wild-type NE. These data are discussed in terms of models of Met ligand properties and proton dependence. [ABSTRACT FROM AUTHOR]

Expression of the truncated (lacking an N-terminal signal sequence) structural gene of Thermus thermophilus cytochrome C552 in the cytoplasm of Escherichia coli yields both dimeric (rC557) and monomeric (rC552) cytochrome c-like proteins [Keightley, J. A., et al. (1998) J. Biol. Chem. 273, 12006-12016], which form spontaneously without the involvement of cytochrome c maturation factors. Cytochrome rC557 is comprised of a dimer and has been structurally characterized [McRee, D., et al. (2001) J. Biol. Chem. 276,6537-6544]. Unexpectedly, the monomeric rC552 transforms spontaneously to a cytochrome- like chromophore having, in its reduced state, the Q00 transition (α-band) at 572 nm (therefore called p572). The X-ray crystallographic structure of rC552, at 1.41 Å resolution, shows that the 2-vinyl group of heme ring I is converted to a [heme-CO-CH2-S-CH2-Ca] conjugate with cysteine 11. Electron density maps obtained from isomorphous crystals of p572 at 1.61 Å resolution reveal that the 2-vinyl group has been oxidized to a formyl group. This explains the lower energy of the Q00 transition, the presence of a new, high-frequency band in the resonance Raman spectra at 1666 cm-1 for oxidized and at 1646 cm-1 for reduced samples, and the greatly altered, paramagnetically shifted ¹H NMR spectrum observed for this species. The overall process defines a novel mechanism for oxidation of the 2-vinyl group to a 2-formyl group and adds to the surprising array of chemical reactions that occur in the interaction of heme with the CXXCH sequence motif in apocytochromes c. [ABSTRACT FROM AUTHOR]

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c551 dynamics based on 15N R1, 15N R2, and {1H}-15N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (=0.924±0.005) and displays little variation in picosecond–nanosecond time scale dynamics over the structure. The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content. Also reported are protection factors calculated from hydrogen-exchange rates. These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions. These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (=0.80±0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3. This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0401-z. On that page (frame on the left side), a link takes you directly to the supplementary material. [ABSTRACT FROM AUTHOR]