Heme proteins—Diversity in structural characteristics,function, and folding

The characteristics of heme prosthetic groups and their binding sites have been analyzed in detail in a data set of nonhomologous heme proteins. Variations in the shape, volume, and chemical composition of the binding site, in the mode of heme binding and in the number and nature of heme–protein interactions are found to result in significantly different heme environments in proteins with different functions in biology. Differences are also seen in the properties of the apo states of the proteins. The apo states of proteins that bind heme permanently in their functional form show some disorder, ranging from local unfolding in the heme binding pocket to complete unfolding to give a random coil. In contrast, proteins that bind heme transiently are fully folded in their apo and holo states, presumably allowing both apo and holo forms to remain biologically active resisting aggregation or proteolysis. The principles identified here provide a framework for the design of de novo proteins that will exhibit tight heme ligand binding and for the identification of the function of structural genomic target proteins with heme ligands. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

An Analysis of the Electron Spin Resonance of Low Spin Ferric Heme Compounds

The electron spin resonance spectra of low spin ferric heme proteins are calculated and compared with experimental spectra recently obtained for a large number of heme proteins. On the basis of observed g values, these heme proteins have been categorized into five prototypes. Since electron spin resonance spectra are quite sensitive to small changes in the local environment about the paramagnetic ion, we have explored in some detail, the possibility that the g value variation in these different types of proteins is due to systematic environmental changes. To this end, we have calculated the g values as a function of tetragonal and rhombic distortions and compared our results with observed behavior. In addition we have also calculated the energy intervals between the low spin states in zero magnetic field and the temperature dependent magnetic moments and studied the effect on these properties also of small symmetry changes.     

Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors

Soluble guanylate cyclase (sGC) is a nitric oxide (NO) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. This family of heme proteins has been named the H-NOX domain, for Heme-Nitric oxide/OXygen binding domain. The key observation from initial studies in this family is that some members, those proteins from most eukaryotes and facultative aerobic prokaryotes, bind NO in a five-coordinate heme complex, but do not bind oxygen (O(2)), the same ligand binding characteristics as sGC. H-NOX family members from obligate aerobic prokaryotes bind O(2) and NO in six-coordinate complexes, similar to the globins and other O(2)-sensing heme proteins. The molecular factors that contribute to these differences in ligand specificity, within a family of sequence related proteins, are the subject of this review.     

Can the axial ligand strength be monitored through spectroscopic measurements?

 The influence of the properties of the axial ligands in heme proteins on the electronic structure of the iron(III) ion has been studied through angular overlap calculations. In particular, a correlation between (a) ligand field parameters and spectroscopic data and (b) the reduction potentials of the iron ion is proposed. The results of this approach are discussed with respect to their relevance for the biological function of different heme proteins. Received and accepted: 7 May 1996     

Phospholipid assisted folding of a denatured heme protein: effect of phosphatidylethanolamine

The role of the aminophospholipid, phosphatidylethanolamine (PE), has been well established to act as a non-protein molecular chaperone in the folding and assembly of polytopic membrane proteins. However, such studies with soluble proteins have not been done so far and in particular with the heme proteins. We have used the heme enzyme, horseradish peroxidase (HRP), as the model heme protein and studied the effect of different phospholipids on its refolding from denatured state. Dimyristoylphosphatidylethanolamine (DMPE), a bilayer-forming PE, was able to increase the reactivation yield of denatured HRP upon 30min refolding at 25 degrees C. However, dioleoylphosphatidylethanolamine (DOPE), containing one double bond in the fatty acid chains, which does not favour bilayer organization, did not support proper refolding. The phospholipids with N-methylated head groups, phosphatidylcholines, e.g., DMPC and DOPC showed differential effects when DMPC remained mostly non-supportive while DOPC on the contrary led to inhibition of the refolding of the denatured heme enzyme. Fluorescence spectroscopic studies also indicated changes in the microenvironments of the heme moiety and the single tryptophan residue of HRP in presence of the aminophospholipid.     

Myoglobin structure and regulation of solvent accessibility of heme pocket

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.     

Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries

We previously reported the de novo design of combinatorial libraries of proteins targeted to fold into four-helix bundles. The sequences of these proteins were designed using a binary code strategy in which each position in the linear sequence is designated as either polar or nonpolar, but the exact identity of the amino acid at each position is varied combinatorially. We subsequently reported that approximately half of these binary coded proteins were capable of binding heme. These de novo heme-binding proteins showed CO binding characteristics similar to natural heme proteins, and several were active as peroxidases. Here we analyze the midpoint reduction potentials and heme binding stoichiometries of several of these de novo heme proteins. All the proteins bound heme with a 1:1 stoichiometry. The reduction potentials ranged from -112 to -176 mV. We suggest that this represents an estimate of the default range of potentials for heme proteins that have neither been prejudiced by rational design nor selected by evolution.     

Structural homology of cytochromes c.

Cytochromes c from many eukaryotic and diverse prokaryotic organisms have been investigated and compared using high-resolution nuclear magnetic resonance spectroscopy. Resonances have been assigned to a large number of specific groups, mostly in the immediate environment of the heme. This information, together with sequence data, has allowed a comparison of the heme environment and protein conformation for these cytochromes. All mitochondrial cytochromes c are found to be very similar to the cytochromes c2 from Rhodospirillaceae. In the smaller bacterial cytochromes, Pseudomonas aeruginosa cytochrome c551 and Euglena gracilis cytochrome c552, the orientation of groups near the heme is very similar, but the folding of the polypeptide chain is different. The heme environment of these two proteins is similar to that of the larger bacterial and mitochondrial cytochromes. Two low-potential cytochromes, Desulfovibrio vulgaris cytochrome c553 and cytochrome c554 from a halotolerant micrococcus have heme environments which are not very similar to those of the other proteins reported here.     

pH dependence of heme electrochemistry in cytochromes investigated by multiconformation continuum electrostatic calculations

Cytochromes belong to a diverse family of heme-containing redox proteins that function as intermediaries in electron transfer chains. They can be soluble, extrinsic, or intrinsic membrane proteins, and are found in different structural motifs (globin, 4-helix bundles, alpha beta roll, beta sandwich). Measured electrochemical midpoint potentials vary over a wide range even though the basic redox reaction at the heme is the same for all cytochromes. The perturbation of the heme electrochemistry is induced by the protein structure. Also, the pH dependence varies since it depends on the strength of interaction between the heme and surrounding residues as well as the ionization states of these groups. Multiconformation continuum electrostatics (MCCE) has been used to investigate the pH dependence of heme electrochemistry in cytochromes with different folds. Often propionates are the primary contributors for pH dependence especially if they are partially protonated in the reduced heme as it is shown for globin cytochrome c551 P. aeruginosa and cytochrome b5 R. norvegicus (alpha beta roll). However, if the propionates are already fully ionized at a certain pH they do not contribute to the pH dependence even if they have big interaction with the heme. At pH 7 there is no propionate contribution for cytochrome f C. reinhardtii (beta sandwich) and the 4-helix bundle c' R. palustris. Other residues can also change their ionization significantly during heme oxidation and therefore be involved in proton release and pH dependence. These residues have been identified for different cytochrome types.     

Ultrafast dynamics of ligands within heme proteins

Physiological bond formation and bond breaking events between proteins and ligands and their immediate consequences are difficult to synchronize and study in general. However, diatomic ligands can be photodissociated from heme, and thus in heme proteins ligand release and rebinding dynamics and trajectories have been studied on timescales of the internal vibrations of the protein that drive many biochemical reactions, and longer. The rapidly expanding number of characterized heme proteins involved in a large variety of functions allows comparative dynamics-structure-function studies. In this review, an overview is given of recent progress in this field, and in particular on initial sensing processes in signaling proteins, and on ligand and electron transfer dynamics in oxidases and cytochromes.     

Ultrafast dynamics of ligands within heme proteins

Physiological bond formation and bond breaking events between proteins and ligands and their immediate consequences are difficult to synchronize and study in general. However, diatomic ligands can be photodissociated from heme, and thus in heme proteins ligand release and rebinding dynamics and trajectories have been studied on timescales of the internal vibrations of the protein that drive many biochemical reactions, and longer. The rapidly expanding number of characterized heme proteins involved in a large variety of functions allows comparative dynamics-structure-function studies. In this review, an overview is given of recent progress in this field, and in particular on initial sensing processes in signaling proteins, and on ligand and electron transfer dynamics in oxidases and cytochromes.     

Iron and heme utilization in Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with the initiation and progression of adult periodontal disease. Iron is utilized by this pathogen in the form of heme and has been shown to play an essential role in its growth and virulence. Recently, considerable attention has been given to the characterization of various secreted and surface-associated proteins of P. gingivalis and their contribution to virulence. In particular, the properties of proteins involved in the uptake of iron and heme have been extensively studied. Unlike other Gram-negative bacteria, P. gingivalis does not produce siderophores. Instead it employs specific outer membrane receptors, proteases (particularly gingipains), and lipoproteins to acquire iron/heme. In this review, we will focus on the diverse mechanisms of iron and heme acquisition in P. gingivalis. Specific proteins involved in iron and heme capture will be described. In addition, we will discuss new genes for iron/heme utilization identified by nucleotide sequencing of the P. gingivalis W83 genome. Putative iron- and heme-responsive gene regulation in P. gingivalis will be discussed. We will also examine the significance of heme/hemoglobin acquisition for the virulence of this pathogen.     

The effect of ligand dynamics on heme electronic transition band III in myoglobin.

Band III is a near-infrared electronic transition at ~13,000 cm(-1) in heme proteins that has been studied extensively as a marker of protein conformational relaxation after photodissociation of the heme-bound ligand. To examine the influence of the heme pocket structure and ligand dynamics on band III, we have studied carbon monoxide recombination in a variety of myoglobin mutants after photolysis at 3 K using Fourier transform infrared temperature-derivative spectroscopy with monitoring in three spectral ranges, (1) band III, the mid-infrared region of (2) the heme-bound CO, and (3) the photodissociated CO. Here we present data on mutant myoglobins V68F and L29W, which both exhibit pronounced ligand movements at low temperature. From spectral and kinetic analyses in the mid-infrared, a small number of photoproduct populations can be distinguished, differing in their distal heme pocket conformations and/or CO locations. We have decomposed band III into its individual photoproduct contributions. Each photoproduct state exhibits a different "kinetic hole-burning" (KHB) effect, a coupling of the activation enthalpy for rebinding to the position of band III. The analysis reveals that the heme pocket structure and the photodissociated CO markedly affect the band III transition. A strong kinetic hole-burning effect results only when the CO ligand resides in the docking site on top of the heme group. Migration of CO away from the heme group leads to an overall blue shift of band III. Consequently, band III can be used as a sensitive tool to study ligand dynamics after photodissociation in heme proteins.     

Expression of prokaryotic and eukaryotic cytochromes c in Escherichia coli

C-type cytochromes from various sources show substantial structural conservation. For the covalent attachment of heme groups to apocytochromes, however, three different enzyme systems have been described so far. We have examined the ability of the heme ligation systems of Escherichia coli and of Saccharomyces cerevisiae to process cytochromes from S. cerevisiae, Paracoccus denitrificans, and Synechocystis sp. PCC 6803. E. coli's maturation system with at least eight different proteins accepted all these cytochromes for heme ligation. The single subunit heme lyase from S. cerevisiae mitochondria, on the other hand, failed to attach heme groups to cytochromes of prokaryotic origin.     

The quantum mechanically mixed-spin state in a non-symbiotic plant hemoglobin: the effect of distal mutation on AHb1 from Arabidopsis thaliana

Non-symbiotic hemoglobins are hexacoordinated heme proteins found in all plants. To gain insight into the importance of the heme hexacoordination and the coordinated distal histidine in general for the possible physiological functions of these proteins, the distal His(E7) of Arabidopsis thaliana hemoglobin (AHb1) was substituted by a leucine residue. The heme properties of the wild-type and mutant proteins have been characterized by electronic absorption, resonance Raman and electron paramagnetic resonance spectroscopic studies at room and low temperatures. Significant differences between the wild-type and mutant proteins have been detected. The most striking is the formation of an uncommon quantum mechanically mixed-spin heme species in the mutant. This is the first observation of such a spin state in a plant hemoglobin. The proportion of this species, which at room temperature coexists with a minor pentacoordinated high-spin form, increases markedly at low temperature.     

Cytochrome c biogenesis in mitochondria--Systems III and V

In c-type cytochromes, heme becomes covalently attached to the polypeptide chain by a reaction between the vinyl groups of the heme and cysteine thiols from the protein. There are two such cytochromes in mitochondria: cytochrome c and cytochrome c(1). The heme attachment is a post-translational modification that is catalysed by different biogenesis proteins in different organisms. Three types of biogenesis system are found or predicted in mitochondria: System I (the cytochrome c maturation system); System III (termed holocytochrome c synthase (HCCS) or heme lyase); and System V. This review focuses primarily on cytochrome c maturation in mitochondria containing HCCS (System III). It describes what is known about the enzymology and substrate specificity of HCCS; the role of HCCS in human disease; import of HCCS into mitochondria; import of apocytochromes c and c(1) into mitochondria and the close relationships with HCCS-dependent heme attachment; and the role of the fungal cytochrome c biogenesis accessory protein Cyc2. System V is also discussed; this is the postulated mitochondrial cytochrome c biogenesis system of trypanosomes and related organisms. No cytochrome c biogenesis proteins have been identified in the genomes of these organisms whose c-type cytochromes also have a unique mode of heme attachment.     

Comparison of the evolution of heme binding and NAD binding proteins.

Of the 85 three-dimensionally characterized residues of cytochrome b5, 51 are structurally and topologically equivalent to the globin fold. When these proteins have been superimposed, the heme irons are found to be less than 1.4 A separated and the heme normals are inclined by less than 9.5 degrees. Comparison of minimum base changes per codon between heme binding and NAD binding proteins are of the same order.     

Ligand dynamics in an electron transfer protein. Picosecond geminate recombination of carbon monoxide to heme in mutant forms of cytochrome c

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to approximately 70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.     

Structural and biochemical characterization of DHC2, a novel diheme cytochrome c from Geobacter sulfurreducens

Multiheme cytochromes c constitute a widespread class of proteins with essential functions in electron transfer and enzymatic catalysis. Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs. Understanding the significance of these motifs is crucial for the elucidation of the highly optimized properties of multiheme cytochromes c, but their spectroscopic investigation is often hindered by the large number and efficient coupling of the individual centers and the limited availability of recombinant protein material. We have identified a diheme cytochrome c, DHC2, from the metal-reducing soil bacterium Geobacter sulfurreducens and determined its crystal structure by the method of multiple-wavelength anomalous dispersion (MAD). The two heme groups of DHC2 pack into one of the typical heme interaction motifs observed in larger multiheme cytochromes, but because of the absence of further, interfering cofactors, the properties of this heme packing motif can be conveniently studied in detail. Spectroscopic properties (UV-vis and EPR) of the protein are typical for cytochromes containing low-spin Fe(III) centers with bis-histidinyl coordination. Midpoint potentials for the two heme groups have been determined to be -135 and -289 mV by potentiometric redox titrations. DHC2 has been produced by recombinant expression in Escherichia coli using the accessory plasmid pEC86 and is therefore accessible for systematic mutational studies in further investigating the properties of heme packing interactions in cytochromes c.