Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.     

ESR characteristics of Photosystem I in deuterium oxide: Further evidence that electron acceptor A1 is a quinone

The appearance of ESR signals from Photosystem I (PS I) electron acceptors A₁ and A₀ in water or deuterium oxide suspension was followed using a low-temperature photoaccumulation technique. In deuterated samples the A₁ signal was narrowed by a factor of 0.66 compared with the control. This effect was fully reversible upon resuspension of treated samples in H₂O. The narrow ESR signal from deuterated A₁ had similar power saturation characteristics to the normal signal; however, a signal from a second component resolved by deuteration was saturated at higher microwave powers than the control. The power saturation behaviour of A⁻₁ in un-modified reaction centres indicated that it is an anionic semiquinone in a ‘protic’ environment. Deuteration reversibly modified the relative extents of reduction of iron sulphur electron acceptors A and B such that centre B became the more stable electron acceptor. The g-value and line-width of iron sulphur centre X was not modified by deuteration although it appeared to become more efficiently reduced. These results are discussed in the light of current evidence from optical, electron spin polarisation and extraction experiments that suggest that A₁ is a quinone, probably vitamin K-1.     

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.     

Interaction of sodium dodecyl sulfate with human native and cross-linked hemoglobins: a transient kinetic study

The interaction of sodium dodecyl sulfate (SDS) at a concentration range (0-515 microM) below the critical micelle concentration (CMC approximately 0.83 mM) with human native and cross-linked oxyhemoglobin (oxyHb) and methemoglobin (metHb) has been investigated by optical spectroscopy and stopped-flow transient kinetic measurements. It is observed that the interaction of SDS with human native and cross-linked oxyHb shows the disappearance of the bands of oxyHb at 541 and 576 nm and the appearance at 537 nm. The resultant spectra are characteristic of low spin (Fe(3+)) hemichrome. Similarly SDS has been found to convert human native and cross-linked high spin (Fe(3+)) metHb to low spin (Fe(3+)) hemichrome. The interaction of SDS with oxyHb suggests a conformational change of the protein in the heme pocket, which may induce the binding of distal histidine to iron leading to the formation of superoxide radical. The formation of hemichrome from metHb is found to be concentration-dependent with SDS. The stopped flow transient kinetic measurements of the interaction of SDS with metHb show that at least four molecules of SDS interact with one molecule of metHb. The interaction of SDS with human cross-linked oxy and met hemoglobin shows results similar to those for human native oxy and met hemoglobin indicating that the covalent modification does not alter the interaction of SDS with cross-linked hemoglobin.     

Crystal structure and spin crossover studies on bis(2,6-bis(benzimidazol-2-yl)pyridine) iron(II) perchlorate

The structure of the [Fe(bzimpy)₂](ClO₄)₂·xH₂O system (x = 0.25) was determined by single crystal X-ray structure analysis. The Fe(II) ion is hexacoordinated by six donor nitrogen atoms. The magnetic properties of the complex were investigated by powder magnetic susceptibility measurements and ESR. The freshly prepared sample does not show any traces of iron(III) impurities but these are formed as a function of time. After 1 year the sample contains 8.2% iron(III) as shown by UV spectroscopy and indicated by g_eff = 4.3 and 2.0 in its ESR spectrum. This explains the recorded ξ versus T behaviour at low temperature: with increasing temperature the ξ value decreases according to the Curie-Weiss law for a S = 5/2 system having an effective g = 4.3. Above 220 K a continuous increase in the ξ value is observed and a spin crossover applies. The spin transition is not complete at room temperature. A pronounced hysteresis is observed upon heating/cooling the sample between 220 and 414 K on the basis of magnetic data and infrared spectra.     

Iron coordination geometry in full-length, truncated, and dehydrated forms of human tyrosine hydroxylase studied by Mössbauer and X-ray absorption spectroscopy

Full-length human tyrosine hydroxylase 1 (hTH1) and a truncated enzyme lacking the 150 N-terminal amino acids were expressed in Escherichia coli and purified either with or without (6×histidine) N-terminal tags. After reconstitution with ⁵⁷Fe(II), the Mössbauer and X-ray absorption spectra of the enzymes were compared before and after dehydration by lyophilization. Before dehydration, >90% of the iron in hTH1 had Mössbauer parameters typical for high-spin Fe(II) in a six-coordinate environment [isomer shift δ(1.8–77?K)=1.26–1.24?mm s^–1 and quadrupole splitting ΔE _Q=2.68?mm s^–1]. After dehydration, the Mössbauer spectrum changed and 63% of the area could be attributed to five-coordinate high-spin Fe(II) (δ=1.07?mm s^–1 and ΔE _Q=2.89?mm s^–1 at 77?K), whereas 28% of the iron remained as six-coordinate high-spin Fe(II) (δ=1.24?mm s^–1 and ΔE _Q=2.87?mm s^–1 at 77?K). Similar changes upon dehydration were observed for truncated TH either with or without the histidine tag. After rehydration of hTH1 the spectroscopic changes were completely reversed. The X-ray absorption spectra of hTH1 in solution and in lyophilized form, and for the truncated protein in solution, corroborate the findings derived from the Mössbauer spectra. The pre-edge peak intensity of the protein in solution indicates six-coordination of the iron, while that of the dehydrated protein is typical for a five-coordinate iron center. Thus, the active-site iron can exist in different coordination states, which can be interconverted depending on the hydration state of the protein, indicating the presence or absence of a water molecule as a coordinating ligand to the iron. The present study explains the difference in iron coordination determined by X-ray crystallography, which has shown a five-coordinate iron center in rat TH, and by our recent spectroscopic study of human TH in solution, which showed a six-coordinated iron center.     

Heme environment in aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis

Resonance Raman spectra have been measured to characterize the heme environment in aldoxime dehydratase (OxdA), a novel hemoprotein, which catalyzes the dehydration of aldoxime into nitrile. The spectra showed that the ferric heme in the enzyme is six-coordinate low spin, whereas the ferrous heme is five-coordinate high spin. We assign a prominent vibration that occurs at 226 cm(-1) in the ferrous enzyme to the Fe-proximal histidine stretching vibration. In the CO-bound form of OxdA, the correlation between the Fe-CO stretching (512 cm(-1)) and C-O stretching (1950 cm(-1)) frequencies also supports our assignment of proximal histidine coordination.     

The heme complex of Hmu O, a bacterial heme degradation enzyme from Corynebacterium diphtheriae. Structure of the catalytic site.

Hmu O, a heme degradation enzyme in Corynebacterium diphtheriae, forms a stoichiometric complex with iron protoporphyrin IX and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Using a multitude of spectroscopic techniques, we have determined the axial ligand coordination of the heme-Hmu O complex. The ferric complex shows a pH-dependent reversible transition between a water-bound hexacoordinate high spin neutral pH form and an alkaline form, having high spin and low spin states, with a pK(a) of 9. (1)H NMR, EPR, and resonance Raman of the heme-Hmu O complex establish that a neutral imidazole of a histidine residue is the proximal ligand of the complex, similar to mammalian heme oxygenase. EPR of the deoxy cobalt porphyrin IX-Hmu O complex confirms this proximal histidine coordination. Oxy cobalt-Hmu O EPR reveals a hydrogen-bonding interaction between the O(2) and an exchangeable proton in the Hmu O distal pocket and two distinct orientations for the bound O(2). Mammalian heme oxygenase has only one O(2) orientation. This difference and the mixed spin states at alkaline pH indicate structural differences in the distal environment between Hmu O and its mammalian counterpart.     

Evidence that electron-dense bodies in Cyanidium caldarium have an iron-storage role

The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs. Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm. Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0. The electron spin resonance (ESR) spectrum of the intact C. caldarium cells showed an isotropic signal at a g value of 2.00. Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal. EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA. The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal. Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.     

Resonance Raman studies of iron spin and axial coordination in distal pocket mutants of ferric myoglobin

We have used resonance Raman spectroscopy to study 11 distal pocket mutants and the "wild type" and native ferric sperm whale myoglobin. The characteristic Raman core-size markers v4, v3, v2, and v10 are utilized to assign the spin and coordination state of each sample. It is demonstrated that replacements of the distal and proximal histidines can discriminate against H2O as a sixth ligand and favor a pentacoordinate Fe3+ atom. Soret absorption band blueshifts are correlated with the pentacoordinate heme environment. One E7 replacement (Arg) leads to an iron spin state change and produces a low spin species. The Glu and Ala mutations at position E11 leave the protein's spin and coordination unaltered. A laser-induced photoreduction effect is observed in all pentacoordinate mutants and seems to be correlated with the loss of the heme-bound water molecule.     

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands

The role of the proximal heme iron ligand in activation of hydrogen peroxide and control of spin state and coordination number in heme proteins is not yet well understood. Although there are several examples of amino acid sidechains with oxygen atoms which can act as potential heme iron ligands, the occurrence of protein-derived oxygen donor ligation in natural protein systems is quite rare. The sperm whale myoglobin cavity mutant H93G Mb (D. Barrick, Biochemistry 33 (1994) 6546) has its proximal histidine ligand replaced by glycine, a mutation which leaves an open cavity capable of accommodation of a variety of unnatural potential proximal ligands. This provides a convenient system for studying ligand-protein interactions. Molecular modeling of the proximal cavity in the active site of H93G Mb indicates that the cavity is of sufficient size to accommodate benzoate and phenolate in conformations that allow their oxygen atoms to come within binding distance of the heme iron. In addition, benzoate may occupy the cavity in an orientation which allows one carboxylate oxygen atom to ligate to the heme iron while the other carboxylate oxygen is within hydrogen bonding distance of serine 92. The ferric phenolate and benzoate complexes have been prepared and characterized by UV-visible and MCD spectroscopies. The benzoate adduct shows characteristics of a six-coordinate high-spin complex. To our knowledge, this is the first known example of a six-coordinate high-spin heme complex with an anionic oxygen donor proximal ligand. The benzoate ligand is displaced at alkaline pH and upon reaction with hydrogen peroxide. The phenolate adduct of H93G Mb is a five-coordinate high-spin complex whose UV-visible and MCD spectra are distinct from those of the histidine 93 to tyrosine (H93Y Mb) mutant of sperm whale myoglobin. The phenolate adduct is stable at alkaline pH and exhibits a reduced reactivity with hydrogen peroxide relative to that of both native ferric myoglobin, and the exogenous ligand-free derivative of ferric H93G Mb. These observations indicate that the identity of the proximal oxygen donor ligand has an important influence on both the heme iron coordination number and the reactivity of the complex with hydrogen peroxide.     

Functional consequences of the creation of an Asp-His-Fe triad in a 3/3 globin

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.     

Genealogy of mammalian cysteine proteinase inhibitors. Common evolutionary origin of stefins, cystatins and kininogens

Resonance Raman spectra are reported for native horseradish peroxidase (HRP) and cytochrome c peroxidase (CCP) at 290, 77 and 9 K, using 406.7 nm excitation, in resonance with the Soret electronic transition. The spectra reveal temperature-dependent equilibria involving changes in coordination or spin state. At 290 K and pH 6.5, CCP contains a mixture of 5- and 6-coordinate high-spin Fe^III heme while at 9 K the equilibrium is shifted entirely to the 6-coordinate species. The spectra indicate weak binding of H₂O to the heme Pe, consistent with the long distance, 2.4 Å, seen in the crystal structure. At 290 K HRP also contains a mixture of high-spin Fe^III hemes with the 5-coordinate form predominant. At low temperature, a small 6-coordinate high-spin component remains but the 5-coordinate high-spin spectrum is replaced by another which is characteristic either of 6-coordinate low-spin or 5-coordinate intermediate spin heme. The latter species is definitely indicated by previous EPR studies at low temperature. This behavior implies that, in contrast to CCP, the distal coordination site of HRP is only partially occupied by H₂O at any temperature and that lowering the temperature significantly weakens the Fe-proximal imidazole bond. Consistent with this inference, the 77 K spectrum of reduced HRP shows an appreciable fraction of molecules having an Fe-imidazole stretching frequency of 222 cm⁻¹, a value indicating weakened H-bonding of the proximal imidazole.

Resonance Roman spectroscopy Horseradish peroxidase Cytochrome c peroxidase Coordination equilibrium     

Low-frequency vibrations in resonance Raman spectra of horse heart myoglobin. Iron-ligand and iron-nitrogen vibrational modes.

The low-frequency regions (150--700 cm-1) of resonance Raman (RR) spectra of various complexes of oxidized and reduced horse heart myoglobin were examined by use of 441.6-nm excitation. In this frequency range, RR spectra show 10 bands common to all myoglobin derivatives (numbered here for convenience from I to X). Relative intensities of bands IV, V, and X constitute good indicators of the doming state of the heme and, consequently, of the spin state of the iron atom. An additional band is present for several complexes (fluorometmyoglobin, hydroxymetmyoglobin, azidometmyoglobin, and oxymyoglobin). Isotopic substitutions on the exogenous ligands and of the iron atom (56Fe leads to 54Fe) allow us to assign these additional lines to the stretching vibrations of the Fe-sixth ligand bond. Similarly, bands II are assigned to stretching vibrations of the Fe-N-(pyrrole) bonds. An assignment of bands VI to stretching vibrations of the Fe-Nepsilon(proximal histidine) bonds is also proposed. Mechanisms for the resonance enhancement of the main low-frequency bands are discussed on the basis of the excitation profiles and of the dispersion curves for depolarization ratios obtained for fluorometmyoglobin and hydroxymetmyoglobin.     

Interaction of rabbit hemopexin with rose bengal and photooxidation of the rose bengal-hemopexin complex.

Rabbit hemopexin associates with rose bengal producing a hypochromic shift in the absorption spectrum of the dye; the extinction coefficient of the dye bound to heme-saturated hemopexin is approximately 20% lower than that of the dye bound to the apoprotein. The interaction of apo- and heme-saturated hemopexin with rose bengal was studied in detail by difference spectroscopy. Apo-hemopexin has one tight binding site for the dye with a dissociation constant in the micromolar range and a set of several weaker binding sites. In contrast, heme-saturated hemopexin has a very low affinity for the dye. Evidence that histidine residues of hemopexin participate in the binding of heme was obtained by photooxidation of hemopexin sensitized by rose bengal. Progressive modification of the 16 histidine residues of hemopexin is effected by illumination of the dye-hemopexin complexes. The midpoint of this pH-dependent reaction is at pH 6.8 +/- 0.1. In 15 min of irradiation, apo-hemopexin loses 50% of its ability to form a low spin hemichrome complex with deuteroheme while only 10% of the ligand coordination to heme iron of the deuteroheme-hemopexin is lost. At that time, approximately 2 more histidine residues are modified in apo-hemopexin than in deuteroheme-hemopexin, and no change is found in other potentially photolabile amino acid residues. The characteristic circular dichroism positive extremum at 231 nm of hemopexin also was decreased by photooxidation, and the loss was slower in the deuteroheme-hemopexin complex than in the apoprotein. When deuteroporphyrin IX was used as the photosensitizing agent, similar results were obtained.     

A pH-dependent aquomet-to-hemichrome transition in crystalline horse methemoglobin

In 1947, Perutz and co-workers reported that crystalline horse methemoglobin undergoes a large lattice transition as the pH is decreased from 7.1 to 5.4. We have determined the pH 7.1 and 5.4 crystal structures of horse methemoglobin at 1.6 and 2.1 A resolution, respectively, and find that this lattice transition involves a 23 A translation of adjacent hemoglobin tetramers as well as changes in alpha heme ligation and the tertiary structure of the alpha subunits. Specifically, when the pH is lowered from 7.1 to 5.4, the Fe(3+) alpha heme groups (but not the beta heme groups) are converted from the aquomet form, in which the proximal histidine [His87(F8)alpha] and a water molecule are the axial heme ligands, to the hemichrome (bishistidine) form, in which the proximal histidine and the distal histidine [His58(E7)alpha] are the axial heme ligands. Hemichrome formation is coupled to a large tertiary structure transition in the eight-residue segment Pro44(CD2)alpha-Gly51(D7)alpha that converts from an extended loop structure at pH 7.1 to a pi-like helix at pH 5.4. The formation of the pi helix forces Phe46(CD4)alpha out of the alpha heme pocket and into the interface between adjacent hemoglobin tetramers where it participates in crystal lattice contacts unique to the pH 5.4 structure. In addition, the transition from aquomet alpha subunits to bishistidine alpha subunits is accompanied by an approximately 1.2 A movement of the alpha heme groups to a more solvent-exposed position as well as the creation of a solvent channel from the interior of the alpha heme pocket to the outside of the tetramer. These changes and the extensive rearrangement of the crystal lattice structure allow the alpha heme group of one tetramer to make direct contact with an alpha heme group on an adjacent tetramer. These results suggest possible functional roles for hemichrome formation in vivo.     

Direct observation of the methionine residues of cytochrome c by 13C nuclear magnetic resonance spectroscopy.

The two Cepsilon-methyl methionine groups in cytochrome c have been chemically enriched (45%) with 13C. Their 13C NMR signals have been monitored in both the oxidized and reduced states and under various solution conditions. Methionine residue 80 showed characteristic chemical shift positions for the reduced Fe(II) and cyano-Fe(III) forms. No signal for methionine 80 was observed in the oxidized Fe(III) form due to the paramagnetic effect of the iron atom to which it is bonded, but the position of the methionine 65 signal was shifted, indicating that it is sensitive to the change of oxidation state. Two well resolved signals were observed at pH 11 for the Fe(III) form but only one was resolved at pH 2, indicating that while methionine 80 is definitely displaced from the iron atom at alkaline pH, it may not be in acid conditions.     

Iron coordination structures of oxygen sensor FixL characterized by Fe K-edge extended x-ray absorption fine structure and resonance raman spectroscopy.

FixL is a heme-based O(2) sensor protein involved in a two-component system of a symbiotic bacterium. In the present study, the iron coordination structure in the heme domain of Rhizobium meliloti FixLT (RmFixLT, a soluble truncated FixL) was examined using Fe K-edge extended x-ray absorption fine structure (EXAFS) and resonance Raman spectroscopic techniques. In the EXAFS analyses, the interatomic distances and angles of the Fe-ligand bond and the iron displacement from the heme plane were obtained for RmFixLT in the Fe(2+), Fe(2+)O(2), Fe(2+)CO, Fe(3+), Fe(3+)F(-), and Fe(3+)CN(-) states. An apparent correlation was found between the heme-nitrogen (proximal His-194) distance in the heme domain and the phosphorylation activity of the histidine kinase domain. Comparison of the Fe-CO coordination geometry between RmFixLT and RmFixLH (heme domain of RmFixL), based on the EXAFS and Raman results, has suggested that the kinase domain directly or indirectly influences steric interaction between the iron-bound ligand and the heme pocket. Referring to the crystal structure of the heme domain of Bradyrhizobium japonicum FixL (Gong, W., Hao, B., Mansy, S. S., Gonzalez, G., Gilles-Gonzalez, M. A., and Chan, M. K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15177-15182), we discussed details of the iron coordination structure of RmFixLT and RmFixLH in relation to an intramolecular signal transduction mechanism in its O(2) sensing.     

NADPH- and NADH-nitrate reductases from soybean leaves.

Treatment of rabbit hemopexin with bromoacetic acid (BrAc) or with diethylpyrocarbonate (DEP) modified histidine residues and produced a concomitant decrease in the protein's ability to form a low-spin hemichrome complex with deuteroheme (ferrideuteroporphyrin IX). Deuteroheme bound to hemopexin before treatment decreased the extent of inactivation by either reagent. After exposure of deuteroheme-hemopexin to 0.16 m BrAc at pH 6.9 for 120 h, 10–11 of the 16 histidine residues of hemopexin were carboxymethylated, but 90–95% of the deuteroheme-hemopexin complex remained intact. Under the same conditions, 12 histidine residues of apo-hemopexin were carboxymethylated, and 95% of the protein's ability to form its normal hemichrome complex with heme (ferriprotoporphyrin IX) was abolished. The alkylated apo-protein, however, did retain a potential to interact with deuteroheme. The apparent dissociation constants for the complexes of metal-free deuteroporphyrin and deuteroheme with BrAc-treated apo-hemopexin were both about 10^?6m and nearly equal to that of the native deuteroporphyrin-hemopexin complex, as assessed by quenching of tryptophan fluorescence.Approximately 10 histidyl residues of the deuteroheme-hemopexin complex, but only about 4 residues of the apo-protein, were modified by DEP before heme-binding was appreciably affected. The effects of DEP on hemopexin were reversed by hydroxylamine at neutral pH, indicating that ethoxyformylation of histidine residues caused the observed inactivation of hemopexin. This and the results of BrAc treatment suggest that hemopexin contains several easily accessible histidine residues which are not critical for its interaction with heme.The conformation-sensitive positive ellipticity at 231 nm of hemopexin was affected by carboxymethylation and ethoxyformylation. Treatment with BrAc had only a small effect on the intrinsic ellipticity of apo-hemopexin, but eliminated the increase in ellipticity produced by interaction of unmodified hemopexin with heme. Treatment with DEP, on the other hand, decreased both intrinsic and extrinsic ellipticity.These results provide further evidence that the heme-hemopexin complex involves histidyl-heme iron coordination. In addition, they show that formation of the histidyl-heme complex not only greatly enhances the strength of the heme-hemopexin interaction but also is important for triggering conformational changes in the protein.