Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.     

Iron-coordinating tyrosine is a key determinant of NEAT domain heme transfer

In humans, heme iron is the most abundant iron source, and bacterial pathogens such as Staphylococcus aureus acquire it for growth. IsdB of S. aureus acquires Fe(III)-protoporphyrin IX (heme) from hemoglobin for transfer to IsdC via IsdA. These three cell-wall-anchored Isd (iron-regulated surface determinant) proteins contain conserved NEAT (near iron transport) domains. The purpose of this work was to delineate the mechanism of heme binding and transfer between the NEAT domains of IsdA, IsdB, and IsdC using a combination of structural and spectroscopic studies. X-ray crystal structures of IsdA NEAT domain (IsdA-N1) variants reveal that removing the native heme-iron ligand Tyr166 is compensated for by iron coordination by His83 on the distal side and that no single mutation of distal loop residues is sufficient to perturb the IsdA-heme complex. Also, alternate heme-iron coordination was observed in structures of IsdA-N1 bound to reduced Fe(II)-protoporphyrin IX and Co(III)-protoporphyrin IX. The IsdA-N1 structural data were correlated with heme transfer kinetics from the NEAT domains of IsdB and IsdC. We demonstrated that the NEAT domains transfer heme at rates comparable to full-length proteins. The second-order rate constant for heme transfer from IsdA-N1 was modestly affected (< 2-fold) by the IsdA variants, excluding those at Tyr166. Substituting Tyr166 with Ala or Phe changed the reaction mechanism to one with two observable steps and decreased observed rates > 15-fold (to 100-fold excess IsdC). We propose a heme transfer model wherein NEAT domain complexes pass heme iron directly from an iron-coordinating Tyr of the donor protein to the homologous Tyr residues of the acceptor protein.     

Breaking the proximal Fe(II)-N(His) bond in heme proteins through local structural tension: lessons from the heme b proteins nitrophorin 4, nitrophorin 7, and related site-directed mutant proteins

The factors leading to the breakage of the proximal iron-histidine bond in the ferroheme protein soluble guanylate cyclase (sGC) are still a matter of debate. This event is a key mechanism in the sensing of NO that leads to the production of the second-messenger molecule cGMP. Surprisingly, in the heme protein nitrophorin 7 (NP7), we noticed by UV-vis absorbance spectroscopy and resonance Raman spectroscopy that heme reduction leads to a loss of the proximal histidine coordination, which is not observed for the other isoproteins (NP1-4). Structural considerations led to the generation and spectroscopic investigation of site-directed mutants NP7(E27V), NP7(E27Q), NP4(D70A), and NP2(V24E). Spectroscopic investigation of these proteins shows that the spatial arrangement of residues Glu27, Phe43, and His60 in the proximal heme pocket of NP7 is the reason for the weakened Fe(II)-His60 bond through steric demand. Spectroscopic investigation of the sample of NP7 reconstituted with 2,4-dimethyldeuterohemin ("symmetric heme") demonstrated that the heme vinyl substituents are also responsible. Whereas the breaking of the iron-histidine bond is rarely seen among unliganded ferroheme proteins, the breakage of the Fe(II)-His bond upon binding of NO to the sixth coordination site is sometimes observed because of the negative trans effect of NO. However, it is still rare among the heme proteins, which is in contrast to the case for trans liganded nitrosyl model hemes. Thus, the question of which factors determine the Fe(II)-His bond labilization in proteins arises. Surprisingly, mutant NP2(V24E) turned out to be particularly similar in behavior to sGC; i.e., the Fe(II)-His bond is sensitive to breakage upon NO binding, whereas the unliganded form binds the proximal His at neutral pH. To the best of our knowledge, NP2(V24E) is the first example in which the ability to use the His-on ? His-off switch was engineered into a heme protein by site-directed mutagenesis other than the proximal His itself. Steric tension is, therefore, introduced as a potential structural determinant for proximal Fe(II)-His bond breakage in heme proteins.     

Novel heme ligand displacement by CO in the soluble hemophore HasA and its proximal ligand mutants: implications for heme uptake and release

HasASM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasRSM, a specific hemophore outer membrane receptor. Heme iron in HasASM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasASM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasASM, HasASM(His32Ala), and HasASM(His83Ala) exhibit similar spectral features and fall above the line that correlates nuFe-CO and nuC-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasASM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasASM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation

Synechocystis hemoglobin contains an unprecedented covalent bond between a nonaxial histidine side chain (H117) and the heme 2-vinyl. This bond has been previously shown to stabilize the ferric protein against denaturation, and also to affect the kinetics of cyanide association. However, it is unclear why Synechocystis hemoglobin would require the additional degree of stabilization accompanying the His117-heme 2-vinyl bond because it also displays endogenous bis-histidyl axial heme coordination, which should greatly assist heme retention. Furthermore, the mechanism by which the His117-heme 2-vinyl bond affects ligand binding has not been reported, nor has any investigation of the role of this bond on the structure and function of the protein in the ferrous oxidation state. Here we report an investigation of the role of the Synechocystis hemoglobin His117-heme 2-vinyl bond on structure, heme coordination, exogenous ligand binding, and stability in both the ferrous and ferric oxidation states. Our results reveal that hexacoordinate Synechocystis hemoglobin lacking this bond is less stable in the ferrous oxidation state than the ferric, which is surprising in light of our understanding of pentacoordinate Hb stability, in which the ferric protein is always less stable. It is also demonstrated that removal of the His117-heme 2-vinyl bond increases the affinity constant for intramolecular histidine coordination in the ferric oxidation state, thus presenting greater competition for the ligand binding site and lowering the observed rate and affinity constants for exogenous ligands.     

Characterization of the periplasmic heme-binding protein shut from the heme uptake system of Shigella dysenteriae

The heme uptake systems by which bacterial pathogens acquire and utilize heme have recently been described. Such systems may utilize heme directly from the host's hemeproteins or via a hemophore that sequesters and transports heme to an outer membrane receptor and subsequently to the translocating proteins by which heme is further transported into the cell. However, little is known of the heme binding and release mechanisms that facilitate the uptake of heme into the pathogenic organism. As a first step toward elucidating the molecular level events that drive heme binding and release, we have undertaken a spectroscopic and mutational study of the first purified periplasmic heme-binding protein (PBP), ShuT from Shigella dysenteriae. On the basis of sequence identity, the ShuT protein is most closely related to the class of PBPs typified by the vitamin B(12) (BtuF) and iron-hydroxamate (FhuD) PBPs and is a monomeric protein having a molecular mass of 28.5 kDa following proteolytic processing of the periplasmic signaling peptide. ShuT binds one b-type heme per monomer with high affinity and bears no significant homology with other known heme proteins. The resonance Raman, MCD, and UV-visible spectra of WT heme-ShuT are consistent with a five-coordinate high spin heme having an anionic O-bound proximal ligand. Site-directed ShuT mutants of the absolutely conserved Tyr residues, Tyr-94 (Y94A) and Tyr-228 (Y228F), which are found in all putative periplasmic heme-binding proteins, were subjected to UV-visible, resonance Raman, and MCD spectroscopic investigations of heme coordination environment and rates of heme release. The results of these experiments confirmed Tyr-94 as the only axial heme ligand and Tyr-228 as making a significant contribution to the stability of heme-loaded ShuT, albeit without directly interacting with the heme iron.     

Iron oxidation state modulates active site structure in a heme peroxidase

We have previously shown [Badyal, S. K., et al. (2006) J. Biol. Chem. 281, 24512-24520] that the distal histidine (His42) in the W41A variant of ascorbate peroxidase binds to the heme iron in the ferric form of the protein but that binding of the substrate triggers a conformational change in which His42 dissociates from the heme. In this work, we show that this conformational rearrangement also occurs upon reduction of the heme iron. Thus, we present X-ray crystallographic data to show that reduction of the heme leads to dissociation of His42 from the iron in the ferrous form of W41A; spectroscopic and ligand binding data support this observation. Structural evidence indicates that heme reduction occurs through formation of a reduced, bis-histidine-ligated species that subsequently decays by dissociation of His42 from the heme. Collectively, the data provide clear evidence that conformational movement within the same heme active site can be controlled by both ligand binding and metal oxidation state. These observations are consistent with emerging data on other, more complex regulatory and sensing heme proteins, and the data are discussed in the context of our developing views in this area.     

Thermodynamic investigation into the mechanisms of proton-coupled electron transfer events in heme protein maquettes

To study the engineering requirements for proton pumping in energy-converting enzymes such as cytochrome c oxidase, the thermodynamics and mechanisms of proton-coupled electron transfer in two designed heme proteins are elucidated. Both heme protein maquettes chosen, heme b-[H10A24]2 and heme b-[delta7-His]2, are four-alpha-helix bundles that display pH-dependent heme midpoint potential modulations, or redox-Bohr effects. Detailed equilibrium binding studies of ferric and ferrous heme b with these maquettes allow the individual contributions of heme-protein association, iron-histidine ligation, and heme-protein electrostatics to be elucidated. These data demonstrate that the larger, less well-structured [H10A24]2 binds heme b in both oxidation states tighter than the smaller and more well-structured [Delta7-His]2 due to a stronger porphyrin-protein hydrophobic interaction. The 66 mV (1.5 kcal/mol) difference in their heme reduction potentials observed at pH 8.0 is due mostly to stabilization of ferrous heme in [H10A24]2 relative to [delta7-His]2. The data indicate that porphyrin-protein hydrophobic interactions and heme iron coordination are responsible for the Kd value of 37 nM for the heme b-[delta7-His]2 scaffold, while the affinity of heme b for [H10A24]2 is 20-fold tighter due to a combination of porphyrin-protein hydrophobic interactions, iron coordination, and electrostatic effects. The data also illustrate that the contribution of bis-His coordination to ferrous heme protein affinity is limited, <3.0 kcal/mol. The 1H+/1e- redox-Bohr effect of heme b-[H10A24]2 is due to the greater absolute stabilization of the ferric heme (4.1 kcal/mol) compared to the ferrous heme (1.4 kcal/mol) binding upon glutamic acid deprotonation, i.e., an electrostatic response mechanism. The 2H+/1e- redox-Bohr effect observed for heme b-[delta7-His]2 is due to histidine protonation and histidine dissociation of ferrous heme b upon reduction, i.e., a ligand loss mechanism. These results indicate that the contribution of porphyrin-protein hydrophobic interactions to heme affinity is critical to maintaining the heme bound in both oxidation states and eliciting an electrostatic response from these designed heme protein scaffolds.     

X-ray crystallographic study of cyanide binding provides insights into the structure-function relationship for cytochrome cd1 nitrite reductase from Paracoccus pantotrophus

We present a 1.59-A resolution crystal structure of reduced Paracoccus pantotrophus cytochrome cd(1) with cyanide bound to the d(1) heme and His/Met coordination of the c heme. Fe-C-N bond angles are 146 degrees for the A subunit and 164 degrees for the B subunit of the dimer. The nitrogen atom of bound cyanide is within hydrogen bonding distance of His(345) and His(388) and either a water molecule in subunit A or Tyr(25) in subunit B. The ferrous heme-cyanide complex is unusually stable (K(d) approximately 10(-6) m); we propose that this reflects both the design of the specialized d(1) heme ring and a general feature of anion reductases with active site heme. Oxidation of crystals of reduced, cyanide-bound, cytochrome cd(1) results in loss of cyanide and return to the native structure with Tyr(25) as a ligand to the d(1) heme iron and switching to His/His coordination at the c-type heme. No reason for unusually weak binding of cyanide to the ferric state can be identified; rather it is argued that the protein is designed such that a chelate-based effect drives displacement by tyrosine of cyanide or a weaker ligand, like reaction product nitric oxide, from the ferric d(1) heme.     

The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) > His39Cys > His39Ser.     

Peroxidase activity in prostaglandin endoperoxide H synthase-1 occurs with a neutral histidine proximal heme ligand

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) convert arachidonic acid to prostaglandin H(2) (PGH(2)), the committed step in prostaglandin and thromboxane formation. Interaction of peroxides with the heme sites in PGHSs generates a tyrosyl radical that catalyzes subsequent cyclooxygenase chemistry. To study the peroxidase reaction of ovine oPGHS-1, we combined spectroscopic and directed mutagenesis data with X-ray crystallographic refinement of the heme site. Optical and Raman spectroscopy of oxidized oPGHS-1 indicate that its heme iron (Fe(3+)) exists exclusively as a high-spin, six-coordinate species in the holoenzyme and in heme-reconstituted apoenzyme. The sixth ligand is most likely water. The cyanide complex of oxidized oPGHS-1 has a six-coordinate, low-spin ferric iron with a v[Fe-CN] frequency at 445 cm(-)(1); a monotonic sensitivity to cyanide isotopomers that indicates the Fe-CN adduct has a linear geometry. The ferrous iron in reduced oPGHS-1 adopts a high-spin, five-coordinate state that is converted to a six-coordinate, low-spin geometry by CO. The low-frequency Raman spectrum of reduced oPGHS-1 reveals two v[Fe-His] frequencies at 206 and 222 cm(-)(1). These vibrations, which disappear upon addition of CO, are consistent with a neutral histidine (His388) as the proximal heme ligand. The refined crystal structure shows that there is a water molecule located between His388 and Tyr504 that can hydrogen bond to both residues. However, substitution of Tyr504 with alanine yields a mutant having 46% of the peroxidase activity of native oPGHS-1, establishing that bonding of Tyr504 to this water is not critical for catalysis. Collectively, our results show that the proximal histidine ligand in oPGHS-1 is electrostatically neutral. Thus, in contrast to most other peroxidases, a strongly basic proximal ligand is not necessary for peroxidase catalysis by oPGHS-1.     

The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11

Mycobacterium tuberculosis is the causative agent of tuberculosis, which is becoming an increasingly global public health problem due to the rise of drug-resistant strains. While residing in the human host, M. tuberculosis needs to acquire iron for its survival. M. tuberculosis has two iron uptake mechanisms, one that utilizes non-heme iron and another that taps into the vast host heme-iron pool. To date, proteins known to be involved in mycobacterial heme uptake are Rv0203, MmpL3, and MmpL11. Whereas Rv0203 transports heme across the bacterial periplasm or scavenges heme from host heme proteins, MmpL3 and MmpL11 are thought to transport heme across the membrane. In this work, we characterize the heme-binding properties of the predicted extracellular soluble E1 domains of both MmpL3 and MmpL11 utilizing absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopic methods. Furthermore, we demonstrate that Rv0203 transfers heme to both MmpL3-E1 and MmpL11-E1 domains at a rate faster than passive heme dissociation from Rv0203. This work elucidates a key step in the mycobacterial uptake of heme, and it may be useful in the development of anti-tuberculosis drugs targeting this pathway.     

Functions of fluctuation in the heme-binding loops of cytochrome b5 revealed in the process of heme incorporation

Cytochrome b(5) (cyt b(5)) holds heme using two axial histidines, His63 and His39, that are located in the centers of the two heme-binding loops. The previous NMR study on the apo form of cyt b(5) (apocyt b(5)) revealed that the loop including His63 exhibits a larger fluctuation compared to the other loop including His39 [Falzone, C. J., Mayer, M. R., Whiteman, E. L., Moore, C. D., and Lecomte, J. T. (1996) Biochemistry 35, 6519-6526]. To understand the significance of the fluctuation, the heme association and dissociation rates of the two loops were compared using two mutants of cyt b(5) in which one of the axial histidines was replaced with leucine. It was demonstrated that the fluctuating loop possesses a significantly slower heme dissociation rate and a faster heme association rate than the other loop. To further verify the importance of the fluctuating loop, the heme association process of wild-type apocyt b(5) was investigated using optical absorption and CD spectroscopies. It was indicated that the process proceeds through the two pathways, and that the dominant pathway involves the initial coordination of His63 located in the fluctuating loop. The urea concentration dependency of the rate constants revealed that the folding of the fluctuating loop is associated with the coordination of His63. It was suggested that the fluctuation enables the loop to have a larger heme-loop contact in the heme-bound conformation. The fluctuating heme-binding loops might be useful for the artificial design of heme-binding proteins.     

On the role of the axial ligand in heme proteins: a theoretical study

We present a systematic investigation of how the axial ligand in heme proteins influences the geometry, electronic structure, and spin states of the active site, and the energies of the reaction cycles. Using the density functional B3LYP method and medium-sized basis sets, we have compared models with His, His+Asp, Cys, Tyr, and Tyr+Arg as found in myoglobin and hemoglobin, peroxidases, cytochrome P450, and heme catalases, respectively. We have studied 12 reactants and intermediates of the reaction cycles of these enzymes, including complexes with H(2)O, OH(-), O(2-), CH(3)OH, O(2), H(2)O(2), and HO(2)(-) in various formal oxidation states of the iron ion (II to V). The results show that His gives ~0.6 V higher reduction potentials than the other ligands. In particular, it is harder to reduce and protonate the O(2) complex with His than with the other ligands, in accordance with the O(2) carrier function of globins and the oxidative chemistry of the other proteins. For most properties, the trend Cys相似文献   

The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin

Synechocystis sp. PCC 6803 hemoglobin is a cyanobacterial Group I truncated hemoglobin. In the absence of an exogenous ligand, its single heme group is coordinated by His46 (E10, distal) and His70 (F8, proximal). The protein can undergo a post-translational modification by which His117 (H16, in the C-terminal helix) reacts with the heme 2-vinyl group to form a Markownikoff adduct. The new C-N bond prevents heme loss, alters the dynamics of the protein, and influences ligand binding to the heme group. To explore the factors conditioning the formation of the cross-link, variants of the protein that contained an alanine or a leucine at position 46 (E10) were prepared. A double replacement (His46Leu and Tyr22 (B10) to Phe) was also performed to perturb the network of interactions stabilizing bound exogenous ligand. The single and double replacements affected the optical and NMR properties of the globin, each in a different fashion. Heme-protein cross-linking, as promoted by sodium dithionite, was retarded by the replacement of His46, but reactivity was recovered when imidazole or cyanide was used as exogenous ligand. In addition, a significant amount of a second product was systematically obtained when dithionite treatment was performed on the cyanide-bound proteins. This species was identified by NMR spectroscopy to be an adduct to the 4-vinyl group. It was concluded that the specificity and rate of the cross-linking reaction depended critically on the nature of the sixth ligand to the heme iron.     

Deciphering the structural role of histidine 83 for heme binding in hemophore HasA

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.