Sarah E. J. Bowman

The Chemistry and Biochemistry of Heme c: Functional Bases for Covalent Attachment

A discussion of the literature concerning the synthesis, function, and activity of heme c-containing proteins is presented. Comparison of the properties of heme c, which is covalently bound to protein, is made to heme b, which is bound noncovalently. A question of interest is why nature uses biochemically expensive heme c in many proteins when its properties are expected to be similar to heme b. Considering the effects of covalent heme attachment on heme conformation and on the proximal histidine interaction with iron, it is proposed that heme attachment influences both heme reduction potential and ligand-iron interactions.

Heme-protein vibrational couplings in cytochrome c provide a dynamic link that connects the heme-iron and the protein surface

The active site of cytochrome c (Cyt c) consists of a heme covalently linked to a pentapeptide segment (Cys-X-X-Cys-His), which provides a link between the heme and the protein surface, where the redox partners of Cyt c bind. To elucidate the vibrational properties of heme c, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c552, including 13C8-heme–, 13C515N-Met–, and 13C15N-polypeptide (pp)–labeled samples, revealing heme-based vibrational modes in the 200- to 450-cm−1 spectral region. Simulations of the NRVS spectra of H. thermophilus cytochrome c552 allowed for a complete assignment of the Fe vibrational spectrum of the protein-bound heme, as well as the quantitative determination of the amount of mixing between local heme vibrations and pp modes from the Cys-X-X-Cys-His motif. These results provide the basis to propose that heme-pp vibrational dynamic couplings play a role in electron transfer (ET) by coupling vibrations of the heme directly to vibrations of the pp at the protein–protein interface. This could allow for the direct transduction of the thermal (vibrational) energy from the protein surface to the heme that is released on protein/protein complex formation, or it could modulate the heme vibrations in the protein/protein complex to minimize reorganization energy. Both mechanisms lower energy barriers for ET. Notably, the conformation of the distal Met side chain is fine-tuned in the protein to localize heme-pp mixed vibrations within the 250- to 400-cm−1 spectral region. These findings point to a particular orientation of the distal Met that maximizes ET.

Native and Unfolded Cytochrome c-Comparison of Dynamics using 2D-IR Vibrational Echo Spectroscopy

Unfolded vs native CO-coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from Hydrogenobacter thermophilus ( Ht-M61A) are studied by IR absorption spectroscopy and ultrafast 2D-IR vibrational echo spectroscopy of the CO stretching mode. The unfolding is induced by guanidinium hydrochloride (GuHCl). The CO IR absorption spectra for both h-cyt c and Ht-M61A shift to the red as the GuHCl concentration is increased through the concentration region over which unfolding occurs. The spectra for the unfolded state are substantially broader than the spectra for the native proteins. A plot of the CO peak position vs GuHCl concentration produces a sigmoidal curve that overlays the concentration-dependent circular dichroism (CD) data of the CO-coordinated forms of both Ht-M61A and h-cyt c within experimental error. The coincidence of the CO peak shift curve with the CD curves demonstrates that the CO vibrational frequency is sensitive to the structural changes induced by the denaturant. 2D-IR vibrational echo experiments are performed on native Ht-M61A and on the protein in low- and high-concentration GuHCl solutions. The 2D-IR vibrational echo is sensitive to the global protein structural dynamics on time scales from subpicosecond to greater than 100 ps through the change in the shape of the 2D spectrum with time (spectral diffusion). At the high GuHCl concentration (5.1 M), at which Ht-M61A is essentially fully denatured as judged by CD, a very large reduction in dynamics is observed compared to the native protein within the approximately 100 ps time window of the experiment. The results suggest the denatured protein may be in a glassy-like state involving hydrophobic collapse around the heme.

Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium: X-ray Crystallographic and Advanced Electron Paramagnetic Resonance Spectroscopic Analysis

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin-echo envelope modulation and electron-nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed (15)N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His6 site of human calprotectin.

Variation and analysis of second-sphere interactions and axial histidinate character in c-type cytochromes.

The electron-donating properties of the axial His ligand to heme iron in cytochromes c (cyts c) are found to be correlated with the midpoint reduction potential (E(m)) in variants of Hydrogenobacter thermophilus cytochrome c(552) (Ht cyt c(552)) in which mutations have been made in and near the Cys-X-X-Cys-His (CXXCH) heme-binding motif. To probe the strength of the His-Fe(III) interaction, we have measured (13)C nuclear magnetic resonance (NMR) chemical shifts for (13)CN(-) bound to heme iron trans to the axial His in Ht Fe(III) cyt c(552) variants. We observe a linear relationship between these (13)C chemical shifts and E(m), indicating that the His-Fe(III) bond strength correlates with E(m). To probe a conserved hydrogen bonding interaction between the axial His Hdelta1 and the backbone carbonyl of a Pro residue, we measured the pK(a) of the axial His Hdelta1 proton (pK(a(2))), which we propose to relate to the His-Fe(III) interaction, reduction potential, and local electrostatic effects. The observed linear relationship between the axial His (13)Cbeta chemical shift and E(m) is proposed to reflect histidinate (anionic) character of the ligand. A linear relationship also is seen between the average heme methyl (1)H chemical shift and E(m) which may reflect variation in axial His electron-donating properties or in the ruffling distortion of the heme plane. In summary, chemical shifts of the axial His and exogenous CN(-) bound trans to His are shown to be sensitive probes of the His-Fe(III) interaction in variants of Ht cyt c(552) and display trends that correlate with E(m).

Temperature Dependent Equilibrium Native to Unfolded Protein Dynamics and Properties Observed with IR Absorption and 2D IR Vibrational Echo Experiments

Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c(552) from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two-dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature-dependent circular dichroism and the IR absorption area ratio R(A)(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous line width and the center frequency shifts as the temperature is increased. The changes in line width and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature-dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature-independent distribution of structures.

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core 13C NMR

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 = ½) 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 (13)C-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.

Effects of Protein Structure on Iron−Polypeptide Vibrational Dynamic Coupling in Cytochrome c

Cytochrome c (Cyt c) has a heme covalently bound to the polypeptide via a Cys-X-X-Cys-His (CXXCH) linker that is located in the interface region for protein–protein interactions. To determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c-552, and variants M13V, M13V/K22M, and A7F, which have structural modifications that alter the composition or environment of the CXXCH pentapeptide loop. Simulations of the NRVS data indicate that the 150–325 cm–1 region is dominated by NHis–Fe–SMet axial ligand and polypeptide motions, while the 325–400 cm–1 region shows dominant contributions from ν(Fe–NPyr) (Pyr = pyrrole) and other heme-based modes. Diagnostic spectral signatures that directly relate to structural features of the heme active site are identified using a quantum chemistry-centered normal coordinate analysis (QCC-NCA). In particular, spectral features that directly correlate with CXXCH loop stiffness, the strength of the Fe–His interaction, and the degree of heme distortion are identified. Cumulative results from our investigation suggest that compared to the wild type (wt), variants M13V and M13V/K22M have a more rigid CXXCH pentapeptide segment, a stronger Fe–NHis interaction, and a more ruffled heme. Conversely, the A7F variant has a more planar heme and a weaker Fe–NHis bond. These results are correlated to the observed changes in reduction potential between wt protein and the variants studied here. Implications of these results for Cyt c biogenesis and electron transfer are also discussed.

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His3Asp Site.

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca(II) ions on the structure and Ni(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni(II)-bound CP-Ser [oligomer of S100A8(C42S) and S100A9(C3S)] crystals obtained in the absence and presence of Ca(II). We present a 2.1 Å resolution crystal structure of Ni(II)-bound CP-Ser and compare this structure to a reported Ni(II)- and Ca(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) J. Am. Chem. Soc. 139, 8828-8836]. This analysis reveals conformational changes associated with coordination of Ca(II) to the EF-hands of S100A9 and that Ca(II) binding affects the coordination number and geometry of the Ni(II) ion bound to the His3Asp site. In contrast, negligible differences are observed for the Ni(II)-His6 site in the absence and presence of Ca(II). Biochemical studies show that, whereas the His6 site has a thermodynamic preference for Ni(II) over Zn(II), the His3Asp site selects for Zn(II) over Ni(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space.