Heme proteins

... Sam P. de Visser Dr. 1 ,; Joan Selverstone Valentine Prof. ... Heme enzymes, such as cytochrome P450 (CYP 450), peroxidase, catalase, heme oxygenase, and nitric oxide synthase, catalyze a diverse array of important metabolic transformations that require the binding and ...

A B S T R A C T Dehaloperoxidase-hemoglobin is the first hemoglobin identified with biologically-relevant oxidative functions, which include peroxidase, peroxygenase and oxidase activities. Herein we report a study of the protein backbone dynamics of DHP using heteronuclear NMR relaxation methods and molecular dynamics (MD) simulations to address the role of protein dynamics in switching from one function to another. The results show that DHP's backbone helical regions and turns have average order parameters of S 2 = 0.87 ± 0.03 and S 2 = 0.76 ± 0.08, respectively. Furthermore, DHP is primarily a monomer in solution based on the overall tumbling correlation time τ m is 9.49 ± 1.65 ns calculated using the prolate diffusion tensor model in the program relax. A number of amino acid residues have significant R ex using the Lipari-Szabo model-free formalism. , which may experience slow conformational motions on the microseconds-milliseconds time scale according to the model. Caution should be used when the model contains > 4 fitting parameters. The program caver3.0 was used to identify tunnels inside DHP obtained from MD simulation snapshots that are consistent with the importance of the Xe binding site, which is located at the central intersection of the tunnels. These tunnels provide diffusion pathways for small ligands such as O 2 , H 2 O and H 2 O 2 to enter the distal pocket independently of the trajectory of substrates and inhibitors, both of which are aromatic molecules.

The electronic structure of the Fe–O2 center in oxy-hemoglobin and oxy-myoglobin is a long-standing issue in the field of bioinorganic chemistry.  Spectroscopic studies have been complicated by the highly delocalized nature of the porphyrin and calculations      require interpretation of multi-determinant wavefunctions for a highly covalent metal site.  Here, iron L-edge X-ray absorption spectroscopy (XAS), interpreted using a valence bond configuration interaction (VBCI) multiplet model, is applied to directly probe the electronic structure of the iron in the biomimetic Fe–O2 heme complex [Fe(pfp)(1-MeIm)O2] (pfp = meso-tetra(α,α,α,α-o-pivalamidophenyl)porphyrin or TpivPP).  This method allows separate estimates of σ-donor, π-donor, and π-acceptor interactions through ligand to metal charge transfer (LMCT) and metal to ligand charge transfer (MLCT) mixing pathways.  The L-edge spectrum of [Fe(pfp)(1-MeIm)O2] is further compared to those of [FeII(pfp)(1-MeIm)2], [FeII(pfp)], and [FeIII(tpp)(ImH)2]Cl (tpp = meso-tetraphenylporphyrin) which have FeII S = 0, FeII S = 1 and FeIII S = 1/2 ground states, respectively.  These serve as references for the three possible contributions to the ground state of oxy-pfp.  The Fe–O2 pfp site is experimentally determined to have both significant σ-donation and a strong π-interaction of the O2 with the iron, with the latter having implications with respect to the spin polarization of the ground state.

In plants, much like in animals, nitric oxide (NO) has been established as an important gaseous signaling molecule. However, contrary to animal systems, NO-sensitive or NO-responsive proteins that bind NO in the form of a sensor or participating in redox reactions have remained elusive. Here, we applied a search term constructed based on conserved and functionally annotated amino acids at the centers of Heme Nitric Oxide/Oxygen (H-NOX) domains in annotated and experimentally-tested gas-binding proteins from lower and higher eukaryotes, in order to identify candidate NO-binding proteins in Arabidopsis thaliana. The selection of candidate NO-binding proteins identified from the motif search was supported by structural modeling. This approach identified AtLRB3 (At4g01160), a member of the Light Response Bric-a-Brac/Tramtrack/Broad Complex (BTB) family, as a candidate NO-binding protein. AtLRB3 was heterologously expressed and purified, and then tested for NO-response. Spectroscopic data confirmed that AtLRB3 contains a histidine-ligated heme cofactor and importantly, the addition of NO to AtLRB3 yielded absorption characteristics reminiscent of canonical H-NOX proteins. Furthermore, substitution of the heme iron-coordinating histidine at the H-NOX center with a leucine strongly impaired the NO-response. Our finding therefore established AtLRB3 as a NO-interacting protein and future characterizations will focus on resolving the nature of this response.

Recent computational results from our group are reviewed on the mechanisms of enzymatic nitrite reduction to nitric oxide or ammonia, as well as nitric oxide reduction to nitrous oxide or ammonia, with some consideration for the electronic structures of electromeric species such as metal-nitric oxide adducts. Metal centers discussed include heme b, heme d 1 , heme c, non-heme diiron, and copper. non-heme iron proteins involved in oxidative and nitrosative stress. In 2005 he obtained a second PhD degree in chemistry under the direction of Prof. Ionel Haiduc, focused on theoretical studies of small molecule activation by hemes. In 2004 he took on a postdoctoral position in England at the University of Essex under the direction of Profs. Chris Cooper and Michael Wilson, working on hemoglobin-based blood substitutes. In 2006 he returned to the Department of Chemistry in Cluj-Napoca, where he became an associate professor in 2007, teaching bioinorganic chemistry and biochemistry. His current research interests are small molecule activation by metalloproteins and related metal centers, as studied by multidisciplinary approaches. The ISI database indexes 36 of his publications, cited a total of 158 times (self-citations excluded).

It is well documented that extracellular alkalization occurs in plants under the challenges by pathogenic microbes. This may eventually induce the pH-dependent extracellular peroxidase-mediated oxidative burst at the site of microbial challenges. By employing the purified proteins of horseradish peroxidase as a model, we have recently proposed a likely role for free Fe2+ in reduction of ferric enzyme of plant peroxidases into ferrous intermediate and oxygen-bound form of enzyme known as Compound III which may eventually releases superoxide anion radical (O2
-), especially under alkaline condition, possibly contributing to the plant defense mechanism. In the present study, we employed the purified protein of soybean peroxidase (SBP) as an additional model, and examined the changes in the redox status of enzyme accompanying the generation of O2
- in response to Fe2+ under alkaline condition.

Using density functional (DFT) calculations, heterolytic oxygen-oxygen bond cleavage is found to be extremely facile in heme and non-heme ferric-hydrogen peroxide complexes, Fe(III)-OH-OH. These findings question the need to invoke double protonation of the " distal " oxygen atom in Fe(III)-O-OH complexes as the universal mechanism of O-O bond cleavage in biological systems.

The CooA family of proteins are prokaryotic CO-sensing
transcription factors that regulate the expression of genes
involved in the utilization of CO as an energy source. They are
homodimeric proteins that contain two hemes. Each monomer
contains an N-terminal heme-binding domain and a
C-terminal DNA-binding domain. Binding of CO to the heme
leads to activation by a large reorientation of the DNAbinding
domain such that the DNA-binding domain is in
position for specific DNA recognition. The crystal structure of
CooA from Rhodospirillum rubrum [RrCooA; Lanzilotta et
al. (2000), Nature Struct. Biol. 7, 876–880] in the inactive COfree
off-state shows that the N-terminal Pro residue of
monomer A coordinates the heme of monomer B and vice
versa. It now appears that the CO replaces the Pro ligand and
that this change is coupled to the activation process. However,
precisely how the replacement of the Pro ligand by CO results
in structural changes some 25 A ° from the CO-binding site
remains unknown. Here, the structure of a CooA variant from
the thermophilic bacterium Carboxydothermus hydrogenoformans
(ChCooA) is reported in which one monomer is fully
in the on-state. The N-terminal region that is displaced by CO
binding is now positioned between the heme-binding and
DNA-binding domains, which requires movement of the
N-terminus by
20 A ° and thus serves as a bridge between the
two domains that helps to orient the DNA-binding domain in
position for DNA binding.

We use resonant X-ray emission spectroscopy and model calculations to quantify the ligand: heme-Fe energy structure of aqueous myoglobins. For reduced (Fe 2+) and oxidized (Fe 3+) states, the removal or addition of an electron primarily involves charge changes on the ligand-site, and not the Fe-site. The results indicate a finite positive/negative charge-transfer energy Delta between the heme-Fe 3d and ligand valence electronic states for Fe 2+/Fe 3+. Thus, the energy difference between the ligand and Fe 3d states (+ Delta ...

The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

Reported here are DFT descriptions of dioxygen reduction at the active site of cytochrlome cd, nitrite reductase, an enzyme with a histidine-ligated heme active site, known to act as cytochrome oxidase. Also explored is the possibility of nitric oxide reduction by histidine-ligated hemes. The energetics of these two processes correlate well with previous findings on other hemoproteins, and allow further elaboration on the newly proposed "thiolate obstruction" theory [Silaghi-Dumitrescu, Eur. J. Inorg. Chem. 2003, 10481. This theory, orthogonal if not opposed to the classical "thiolate push" dogma [Dawson et a/., J. Am. Chern. Soc. 1976, 98, 3707, argues that efficient reduction of diatomics such as dioxygen and nitric oxide is more readily accomplished by histidineligated hemes than by thiolat* ligated hemes. Generalizing, for reductive processes involving a small diatomic as the sole substrate, neutral ligands (e.g., histidine in cytochrme oxidases and heme oxygenase, lysine in cytochrome c nitrite reductase) are found to always be preferable over anionic ligands. By contrast, in enzymes designed to deal with more than one substrate, anionic ligands are preferable (e.g., cysteine or tyrosine in monooxygenases), since they allow the safety switches needed to avoid uncoupling in their race against entropy. INTRODUCTION Proteins such as cytochrome P450 (P450), horseradish peroxidase (HRP) or hemoglobin are all known to bind andlor reduce (activate) dioxygen at their heme active sites, in the ferrous form.[l-I I] The thiolate-ligated ferrous heme of P450 binds dioxygen and promotes proton-dependent 0-0 bond cleavage following a one-electron reductiin.[l] By contrast, the ferrous histidine-ligated heme of hemoglobin only binds dioxygen in a reversible manner.[l2] Hemoprotein ferriohydroperoxo complexes decay via proton-assisted heterolytic cleavage of the 0-0 bond to yield water and a [ F ~ = o ] ~ ' unit (known as Compound I), where the iron is considered to be in the formal oxidation state +4 and a further oxidizing equivalent is proposed to be delocalized onto the porphyrin. Two active-site "distal" residues, a histidine and an arginine, are crucial in promoting 0-0 bond cleavage in the ferric-hydroperoxo complex of canonical (histidine-ligated) peroxidases. The hemoglobin active site lacks the distal arginine and is much less efficient than HRP at activating peroxide.[l-101 The heme-thiolate active site of P450 does not contain the histidine-arginine catalytic pair of HRP, yet still deaves the 0-0 bond of its ferriohydroperoxo complex

Surface hydrophobicity of leghemoglobin (Lb), myoglobin (Mb) ,cytochrome c (Cyt C) and hemoglobin (Hb) under heated (80 °C for 30 min.) and unheated conditions and at varying pH values (3.0, 5.0, 7.0, and 9.0) were measured using ANS, CPA, and PRODAN. Hydrophobicity was lower at pH 9.0, 5.0 and 7.0 for all proteins measured by PRODAN, for ANS and for CPA respectively. Lb shows maximum hydrophobic nature with ANS and CPA may and less with PRODAN compared to other heme proteins, indicating that Lb favor less ionic interaction. These results suggest that the presence of a permanent charge of fluorescent probes can affect protein hydrophobicity values measured under various pH conditions for Lb.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 is an adaptive and beneficial response in renal and nonrenal settings of tissue injury. The purpose of this study was to characterize the regulation of the human HO-1 gene in renal proximal tubule and aortic endothelial cells in response to heme and cadmium. Evaluation of multiple human HO-1 promoter-reporter constructs up to -9.1 kb demonstrated only a partial response to heme and cadmium. In an effort to mimic endogenous stimulus-dependent levels of HO-1 induction, we evaluated the entire 12.5 kb of the human HO-1 gene, including introns and exons, in conjunction with a -4.5-kb human HO-1 promoter and observed significant heme- and cadmium-mediated induction of the reporter gene, suggesting the presence of an internal enhancer. Enhancer function was orientation independent and required a region between -3.5 and -4.5 kb of the human HO-1 p...

We use resonant X-ray emission spectroscopy and model calculations to quantify the ligand: heme-Fe energy structure of aqueous myoglobins. For reduced (Fe2+) and oxidized (Fe3+) states, the removal or addition of an electron primarily involves charge changes on the ligand-site, and not the Fe-site. The results indicate a finite positive/negative charge-transfer energy Δ between the heme-Fe 3d and ligand valence electronic states for Fe2+/Fe3+. Thus, the energy difference between the ligand and Fe 3d states (+Δ or -Δ) determines the charge properties of myoglobins. The study provides a reliable method for characterizing ligand-metal binding of biological systems in solution.

Ocular neovascularization underlies major blinding eye diseases such as "wet" age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fech(m1Pas) mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therape...

Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O-O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.

Nathalie Hill-Kapturczak, PhD*, Eric Sikorski, PhD*, Christy Voakes*, Jairo Garcia*, Harry S. Nick, PhD#, Anupam Agarwal, MD* ... *Department of Medicine, Division of Nephrology, Hypertension and Transplantation ... #Department of Neuroscience, University of Florida, Gainesville, ...