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

Characterization of anammox hydrazine dehydrogenase, a key N2-producing enzyme in the global nitrogen cycle

Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one out of ten paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAOrelated hydroxylamine-oxidizing enzyme kustc1061 (KsHOX) from K. stuttgartiensis. Interestingly, the HDH trimers formed octamers in solution, each octamer harbouring an amazing 192 c-type heme moieties. While HAO and KsHOX are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well-defined HAO and HOX. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (“P460”) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms

One fold, two functions: cytochrome P460 and cytochrome c′-β from the methanotroph Methylococcus capsulatus (Bath)

Nature is adept at utilising highly similar protein folds to carry out very different functions, yet the mechanisms by which this functional divergence occurs remain poorly characterised. In certain methanotrophic bacteria, two homologous pentacoordinate c-type heme proteins have been identified: a cytochrome P460 (cyt P460) and a cytochrome c′-β (cyt cp-β). Cytochromes P460 are able to convert hydroxylamine to nitrous oxide (N2O), a potent greenhouse gas. This reactivity is similar to that of hydroxylamine oxidoreductase (HAO), which is a key enzyme in nitrifying and methanotrophic bacteria. Cyt P460 and HAO both have unusual protein-heme cross-links, formed by a Tyr residue in HAO and a Lys in cyt P460. In contrast, cyts cp-β (the only known cytochromes c′ with a β-sheet fold) lack this crosslink and appears to be optimized for binding non-polar molecules (including NO and CO) without enzymatic conversion. Our bioinformatics analysis supports the proposal that cyt cp-β may have evolved from cyt P460 via a gene duplication event. Using high-resolution X-ray crystallography, UV-visible absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopy, we have characterized the overall protein folding and active site structures of cyt cp-β and cyt P460 from the obligate methanotroph, Methylococcus capsulatus (Bath). These proteins display a similar β-sheet protein fold, together with a pattern of changes to the heme pocket regions and localised tertiary structure that have converted a hydroxylamine oxidizing enzyme into a gas-binding protein. Structural comparisons provide insights relevant to enzyme redesign for synthetic enzymology and engineering of gas sensor proteins. We also show the widespread occurrence of cyts cp-β and characterise their phylogeny

Hemin and bile pigments are the secondary structure regulators of intrinsically disordered antimicrobial peptides

The interaction of protoporphyrin compounds of human origin with the major bee venom component melittin (26 a.a., Z +6) and its hybrid derivative (CM15, 15 a.a., Z +6) were studied by a combination of various spectroscopic methods. Throughout a two-state, concentration-dependent process, hemin and its metabolites (biliverdin, bilirubin, bilirubin ditaurate) increase the parallel β-sheet content of the natively unfolded melittin, suggesting the oligomerization of the peptide chains. In contrast, α-helix promoting effect was observed with the also disordered but more cationic CM15. According to fluorescence quenching experiments, the sole Trp residue of melittin is the key player during the binding, in the vicinity of which the first pigment molecule is accommodated presumably making indole-porphyrin π-π stacking interaction. As circular dichroism titration data suggest, cooperative association of additional ligands subsequently occurs, resulting in multimeric complexes with an apparent dissociation constant ranged from 20 to 65 μM. Spectroscopic measurements conducted with the bilirubin catabolite urobilin and stercobilin refer to the requirement of intact dipyrrinone moieties for inducing secondary structure transformations. The binding topography of porphyrin rings on a model parallel β-sheet motif was evaluated by absorption spectroscopy and computational modeling showing a slipped-cofacial binding mode responsible for the red shift and hypochromism of the Soret band. Our results may aid to recognize porphyrin-responsive binding motifs of biologically relevant, intrinsically disordered peptides and proteins, where transient conformations play a vital role in their functions

Designed Metal-ATCUN Derivatives: Redox- and Non-redox-Based Applications Relevant for Chemistry, Biology, and Medicine

UID/QUI/50006/2019The designed "ATCUN'' motif (amino-terminal copper and nickel binding site) is a replica of naturally occurring ATCUN site found in many proteins/peptides, and an attractive platform for multiple applications, which include nucleases, proteases, spectroscopic probes, imaging, and small molecule activation. ATCUN motifs are engineered at periphery by conjugation to recombinant proteins, peptides, fluorophores, or recognition domains through chemically or genetically, fulfilling the needs of various biological relevance and a wide range of practical usages. This chemistry has witnessed significant growth over the last few decades and several interesting ATCUN derivatives have been described. The redox role of the ATCUN moieties is also an important aspect to be considered. The redox potential of designed M-ATCUN derivatives is modulated by judicious choice of amino acid (including stereochemistry, charge, and position) that ultimately leads to the catalytic efficiency. In this context, a wide range of M-ATCUN derivatives have been designed purposefully for various redox- and non-redox-based applications, including spectroscopic probes, target-based catalytic metallodrugs, inhibition of amyloid-beta toxicity, and telomere shortening, enzyme inactivation, biomolecules stitching or modification, next-generation antibiotic, and small molecule activation.publishersversionpublishe

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 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

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