Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c

Cytochrome (cyt) c is an important electron transfer protein. The ruffling deformation of its heme cofactor has been suggested to relate to its electron transfer rate. However, there is no direct experimental evidence demonstrating this correlation. In this work, we studied Pseudomonas aeruginosa cytochrome c551 and its F7A mutant. These two proteins, although similar in their X-ray crystal structure, display a significant difference in their heme outof- plane deformations, mainly along the ruffling coordinate. Resonance Raman and vibrational coherence measurements also indicate significant differences in ruffling-sensitive modes, particularly the low-frequency γa mode found between ~50-60 cm-1. This supports previous assignments of γa as having a large ruffling content. Measurement of the photoreduction kinetics finds an order of magnitude decrease of the photoreduction cross-section in the F7A mutant, which has nearly twice the ruffling deformation as the WT. Additional measurements on cytochrome c demonstrate that heme ruffling is correlated exponentially with the electron transfer rates and suggest that ruffling could play an important role in redox control. A major relaxation of heme ruffling in cytochrome c, upon binding to the mitochondrial membrane, is discussed in this context

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 c 552, including 13C8-heme-, 13C 515N-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-XCys-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

Biological role and applications of covalent heme attachment to polypeptides

Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2014.Heme is a biological cofactor that performs an array of functions, including electron transfer, redox catalysis, and gas sensing and transport. The ligands and local environment of the heme group are essential in tuning the properties of the heme to perform in such diverse roles. A subset of heme cofactors, known as hemes c, are covalently ligated to the protein backbone via a CXXCH peptide motif, and are mainly dedicated to performing electron transfer. The research described in this thesis focuses on the ways in which covalent attachment of heme c tunes heme properties relevant to electron transfer. The heme attachment motif is known to promote an out-of-plane distortion of the heme called ruffling. Variants of bacterial cytochromes c from Hydrogenobacter thermophilus and Pseudomonas aeruginosa in which the magnitude of heme ruffling has been altered were analyzed by their paramagnetic NMR shifts, enabling a full description of the influence of ruffling on heme hyperne shifts. The analysis was then used to determine the influence of the length of the heme attachment motif, which covalently binds the heme, on the extent of heme ruffling. The analysis determined that in H. thermophilus cytochrome c, both longer (CX4CH) and shorter (CX1CH) heme attachment motifs enhance the heme ruffling distortion. Increased heme ruffling, measured by the hyperfine NMR shift analysis, correlates to a decreased redox potential of the heme in a number of cytochrome c variants, suggesting that a biological role of the heme attachment motif may be to tune the redox potential of the heme to lower potentials via the heme ruffling distortion. The vibrational profile of the heme attachment motif was also investigated with nuclear resonance vibrational spectroscopy, with implications for understanding how heme covalent attachment optimizes electron transfer through vibrational coupling. Finally, an application of the covalent attachment of heme to a peptide derived from cytochrome c is developed via substitution of cobalt for the heme iron. The resulting cobalt microperoxidase is demonstrated to be a rare example of a hydrogen-evolving electrocatalyst that functions in neutral water using a non-noble metal

Comparing substrate specificity between cytochrome c maturation and cytochrome c heme lyase systems for cytochrome c biogenesis

Hemes c are characterized by their covalent attachment to a polypeptide via a widely conserved CXXCH motif. There are multiple biological systems that facilitate heme c biogenesis. System I, the cytochrome c maturation (CCM) system, is found in many bacteria and is commonly employed in the maturation of bacterial cytochromes c in Escherichia coli-based expression systems. System III, cytochrome c heme lyase (CCHL), is an enzyme found in the mitochondria of many eukaryotes and is used for heterologous expression of mitochondrial holocytochromes c. To test CCM specificity, a series of Hydrogenobacter thermophilus cytochrome c552 variants was successfully expressed and matured by the CCM system with CXnCH motifs where n = 1-4, further extending the known substrate flexibility of the CCM system by successful maturation of a bacterial cytochrome c with a novel CXCH motif. Horse cytochrome c variants with both expanded and contracted attachment motifs (n = 1-3) were also tested for expression and maturation by both CCM and CCHL, allowing direct comparison of CCM and CCHL substrate specificities. Successful maturation of horse cytochrome c by CCHL with an extended CXXXCH motif was observed, demonstrating that CCHL shares the ability of CCM to mature hemes c with extended heme attachment motifs. In contrast, two single amino acid mutants were found in horse cytochrome c that severely limit maturation by CCHL, yet were efficiently matured with CCM. These results identify potentially important residues for the substrate recognition of CCHL. © The Royal Society of Chemistry

Biological Significance and Applications of Heme c Proteins and Peptides

© 2015 American Chemical Society. ConspectusHemes are ubiquitous in biology and carry out a wide range of functions. The heme group is largely invariant across proteins with different functions, although there are a few variations seen in nature. The most common variant is heme c, which is formed by a post-translational modification in which heme is covalently linked to two Cys residues on the polypeptide via thioether bonds. In this Account, the influence of this covalent attachment on heme c properties and function is discussed, and examples of how covalent attachment has been used in selected applications are presented.Proteins that bind heme c are among the most well-characterized proteins in biochemistry. Most of these proteins are cytochromes c (cyts c) that serve as electron carriers in photosynthesis and respiration. Despite the intense study of cyts c, the functional significance of heme covalent attachment has remained elusive. One observation is that heme c reaches a lower reduction potential in nature than its noncovalently linked counterpart, heme b, when comparing proteins with the same axial ligands. Furthermore, covalent attachment is known to enhance protein stability and allow the heme to be relatively solvent exposed. However, an inorganic chemistry perspective on the effects of covalent attachment has been lacking. Spectroscopic measurements and computations on cyts c and model systems reveal a number of effects of covalent attachment on heme electronic structure and reactivity. One is that the predominant nonplanar ruffling distortion seen in heme c lowers heme reduction potential. Another is that covalent attachment influences the interaction of the heme iron with the proximal His ligand. Heme ruffling also has been shown to influence electronic coupling to redox partners and, therefore, electron transfer rates by altering the distribution of the orbital hole on the porphyrin in oxidized cyt c. Another consequence of heme covalent attachment is the strong vibrational coupling seen between the iron and the protein surface as revealed by nuclear resonance vibrational spectroscopy studies. Finally, heme covalent attachment is proposed to be an important feature supporting multiple roles of cyt c in programmed cell death (apoptosis).Heme covalent attachment is not only vital for the biological functions of cyt c but also provides a useful handle in a number of applications. For one, the engineering of heme c onto an exposed portion of a protein of interest has been shown to provide a visible affinity purification tag. In addition, peptides with covalently attached heme, known as microperoxidases, have been studied as model compounds and oxidation catalysts and, more recently, in applications for energy conversion and storage. The wealth of insight gained about heme c through fundamental studies of cyts c forms a basis for future efforts toward engineering natural and artificial cytochromes for a variety of applications

Hydrogen evolution from neutral water under aerobic conditions catalyzed by cobalt microperoxidase-11

A molecular electrocatalyst is reported that reduces protons to hydrogen (H2) in neutral water under aerobic conditions. The biomolecular catalyst is made from cobalt substitution of microperoxidase-11, a water-soluble heme-undecapeptide derived from the protein horse cytochrome c. In aqueous solution at pH 7.0, the catalyst operates with near quantitative Faradaic efficiency, a turnover frequency ∼6.7 s-1 measured over 10 min at an overpotential of 852 mV, and a turnover number of 2.5 × 10 4. Catalyst activity has low sensitivity to oxygen. The results show promise as a hydrogenase functional mimic derived from a biomolecule. © 2013 American Chemical Society

Biological Significance and Applications of Heme <i>c</i> Proteins and Peptides

ConspectusHemes are ubiquitous in biology and carry out a wide range of functions. The heme group is largely invariant across proteins with different functions, although there are a few variations seen in nature. The most common variant is heme <i>c</i>, which is formed by a post-translational modification in which heme is covalently linked to two Cys residues on the polypeptide via thioether bonds. In this Account, the influence of this covalent attachment on heme <i>c</i> properties and function is discussed, and examples of how covalent attachment has been used in selected applications are presented.Proteins that bind heme <i>c</i> are among the most well-characterized proteins in biochemistry. Most of these proteins are cytochromes <i>c</i> (cyts <i>c</i>) that serve as electron carriers in photosynthesis and respiration. Despite the intense study of cyts <i>c</i>, the functional significance of heme covalent attachment has remained elusive. One observation is that heme <i>c</i> reaches a lower reduction potential in nature than its noncovalently linked counterpart, heme <i>b</i>, when comparing proteins with the same axial ligands. Furthermore, covalent attachment is known to enhance protein stability and allow the heme to be relatively solvent exposed. However, an inorganic chemistry perspective on the effects of covalent attachment has been lacking. Spectroscopic measurements and computations on cyts <i>c</i> and model systems reveal a number of effects of covalent attachment on heme electronic structure and reactivity. One is that the predominant nonplanar ruffling distortion seen in heme <i>c</i> lowers heme reduction potential. Another is that covalent attachment influences the interaction of the heme iron with the proximal His ligand. Heme ruffling also has been shown to influence electronic coupling to redox partners and, therefore, electron transfer rates by altering the distribution of the orbital hole on the porphyrin in oxidized cyt <i>c</i>. Another consequence of heme covalent attachment is the strong vibrational coupling seen between the iron and the protein surface as revealed by nuclear resonance vibrational spectroscopy studies. Finally, heme covalent attachment is proposed to be an important feature supporting multiple roles of cyt <i>c</i> in programmed cell death (apoptosis).Heme covalent attachment is not only vital for the biological functions of cyt <i>c</i> but also provides a useful handle in a number of applications. For one, the engineering of heme <i>c</i> onto an exposed portion of a protein of interest has been shown to provide a visible affinity purification tag. In addition, peptides with covalently attached heme, known as microperoxidases, have been studied as model compounds and oxidation catalysts and, more recently, in applications for energy conversion and storage. The wealth of insight gained about heme <i>c</i> through fundamental studies of cyts <i>c</i> forms a basis for future efforts toward engineering natural and artificial cytochromes for a variety of applications

The influence of heme ruffling on spin densities in ferricytochromes c probed by heme core \u3csup\u3e13\u3c/sup\u3eC 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 = 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. © 2013 American Chemical Society

A weakly coordinating anion as a tripodal Br3 -ligand for platinum(IV) - Structure of [(closo-CB\u3csub\u3e11\u3c/sub\u3eH\u3csub\u3e6\u3c/sub\u3eBr \u3csub\u3e6\u3c/sub\u3e)PtMe\u3csub\u3e3\u3c/sub\u3e]\u3csup\u3e1\u3c/sup\u3e

Synthesis, structure, and NMR spectroscopic data for [(closo-CB H Br )PtMe ] are reported. This neutral platinum(IV) complex contains the closo-CB H Br anion bonded to the trimethylplatinum(IV) cation via three boron-bound bromines. Closo-CB H Br , which often acts as weakly coordinating or even non-coordinating anion, adopts here a role still very rare for this anion: it acts as a tripodal capping ligand enabling a pseudo-octahedral geometry at a d6 metal center. Three bromines from the lower hemisphere of the hexahalogenated carboranate coordinate to Pt(IV), and distortions from ideal octahedral angles at Pt are marginal (\u3c3°). Pt-Br bond lengths are 2.7279(18), 2.7129(17), and 2.7671(18) Å. Using the J coupling constant of Pt-bonded methyl groups (79.0 Hz) as indicator of the donor strength of the tripodal cap, the prediction is obtained that closo-CB H Br is a relatively weak donor toward the trimethylplatinum(IV) cation. Ligand competition equilibria can be expected to depend on both the intrinsic donor strengths of competing ligands and on the effects of charge and geometry. We observe that closo-CB H Br is capable of replacing acetone from Me Pt(acetone) , whereas BF counterion is unable to replace acetone under similar conditions. © 2008 NRC Canada. 11 6 6 3 11 6 6 11 6 6 ptH 11 6 6 11 6 6 3 3 4 - - 2 - - +

Hydrogen Evolution from Neutral Water under Aerobic Conditions Catalyzed by Cobalt Microperoxidase-11

A molecular electrocatalyst is reported that reduces protons to hydrogen (H₂) in neutral water under aerobic conditions. The biomolecular catalyst is made from cobalt substitution of microperoxidase-11, a water-soluble heme-undecapeptide derived from the protein horse cytochrome <i>c</i>. In aqueous solution at pH 7.0, the catalyst operates with near quantitative Faradaic efficiency, a turnover frequency ∼6.7 s^–1 measured over 10 min at an overpotential of 852 mV, and a turnover number of 2.5 × 10⁴. Catalyst activity has low sensitivity to oxygen. The results show promise as a hydrogenase functional mimic derived from a biomolecule