Heme Coordination Structure Research Articles

Characterization of a Cobalt-Substituted Globin-Coupled Oxygen Sensor Histidine Kinase from Anaeromyxobacter sp. Fw109-5: Insights into Catalytic Regulation by Its Heme Coordination Structure.

Heme-based gas sensors are an emerging class of heme proteins. AfGcHK, a globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5, is an oxygen sensor enzyme in which oxygen binding to Fe(II) heme in the globin sensor domain substantially enhances its autophosphorylation activity. Here, we reconstituted AfGcHK with cobalt protoporphyrin IX (Co-AfGcHK) in place of heme (Fe-AfGcHK) and characterized the spectral and catalytic properties of the full-length proteins. Spectroscopic analyses indicated that Co(III) and Co(II)-O2 complexes were in a 6-coordinated low-spin state in Co-AfGcHK, like Fe(III) and Fe(II)-O2 complexes of Fe-AfGcHK. Although both Fe(II) and Co(II) complexes were in a 5-coordinated state, Fe(II) and Co(II) complexes were in high-spin and low-spin states, respectively. The autophosphorylation activity of Co(III) and Co(II)-O2 complexes of Co-AfGcHK was fully active, whereas that of the Co(II) complex was moderately active. This contrasts with Fe-AfGcHK, where Fe(III) and Fe(II)-O2 complexes were fully active and the Fe(II) complex was inactive. Collectively, activity data and coordination structures of Fe-AfGcHK and Co-AfGcHK indicate that all fully active forms were in a 6-coordinated low-spin state, whereas the inactive form was in a 5-coordinated high-spin state. The 5-coordinated low-spin complex was moderately active—a novel finding of this study. These results suggest that the catalytic activity of AfGcHK is regulated by its heme coordination structure, especially the spin state of its heme iron. Our study presents the first successful preparation and characterization of a cobalt-substituted globin-coupled oxygen sensor enzyme and may lead to a better understanding of the molecular mechanisms of catalytic regulation in this family.

Supramolecular Assemblies of C-Type Cytochromes Based on 3D Domain Swapping

We have previously shown that horse cytochrome (cyt) c forms polymers from monomers by 3D domain swapping (herein domain swapping) its C-terminal α-helix successively (runaway domain swapping) [1]. The C-terminal α-helix of dimeric horse cyt c was displaced from its original position in the monomer, and the Met80–heme coordination was perturbed significantly in the dimer, causing higher cyanide ion binding affinity and peroxidase activity compared to those in the monomer. Cyt c formed domain-swapped oligomers by the interaction between the N- and C-terminal α-helices at the early stage of folding from its unfolded state [2], and the interaction important for domain swapping was detected in the monomer molten globule state [3]. However, cyt c domain swapping occurred regardless of Met80 mutation to Ala, and the domain-swapped dimer stability was less affected by the Met80–heme coordination [4]. High-order domain-swapped oligomers of HT cyt c 552 were produced during E. coli culture, whereas the domain-swapped protein amount decreased when the protein stability was decreased by mutation, indicating that exceedingly stable proteins may have disadvantages forming domain-swapped oligomers in cells [5]. Cyt cb 562 also domain swapped and formed a dimer, where the two helices in the N-terminal region of one protomer interacted with the other two helices in the C-terminal region of the other protomer. In the crystal, three domain-swapped cyt cb 562 dimers formed a unique cage structure with a Zn-SO4 cluster inside the cavity [6]. We have previously shown that myoglobin (Mb) forms a domain-swapped dimer with two extended α-helices, and constructed an artificial heterodimeric protein with two different heme active sites (a bis-histidine-coordinated heme and a H2O/histidine-coordinated heme) by domain swapping two Mb surface mutants with opposite charges [7]. In this study, to obtain a c-type cyt heterodimer, we constructed two c-type cyt-based chimeric proteins and domain swapped them. The two chimeric proteins formed a domain-swapped dimer with two His/Met coordinate hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domain-swapped c-type cyt heterodimer with His/Met and His/H2O coordinate hemes was formed [8]. Protein amyloids receive much attention due to their correlation with serious diseases and to their promising mechanical and optical properties as future materials. A single spherical assembly of amyloid fibrils has been shown to generate by laser trapping domain-swapped dimeric cyt c [2]. Amyloid formation dynamics and amyloid-based micro-structure fabrication were demonstrated based on direct observation of a single spherical assembly, which foresees a new approach in amyloid studies.

Rational Design of Domain-Swapping-Based c-Type Cytochrome Heterodimers by Using Chimeric Proteins.

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three-dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c-type cytochrome-based chimeric proteins have been constructed and the domains swapped. According to X-ray crystallographic analysis, the two chimeric proteins formed a domain-swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H2 O coordinated hemes was formed. Binding of an oxygen molecule to the His/H2 O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe-O2 stretching band was observed at 580 cm-1 for the reduced/oxygenated heterodimer (at 554 cm-1 under an 18 O2 atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor AfGcHK.

AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)-SH heme complex was quickly converted into Fe(II) and Fe(II)-O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)-SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)-OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.

Catalytic enhancement of the heme-based oxygen-sensing phosphodiesterase EcDOS by hydrogen sulfide is caused by changes in heme coordination structure.

EcDOS is a heme-based O2-sensing phosphodiesterase in which O2 binding to the heme iron complex in the N-terminal domain substantially enhances catalysis toward cyclic-di-GMP, which occurs in the C-terminal domain. Here, we found that hydrogen sulfide enhances the catalytic activity of full-length EcDOS, possibly owing to the admixture of 6-coordinated heme Fe(III)-SH(-) and Fe(II)-O2 complexes generated during the reaction. Alanine substitution at Met95, the axial ligand for the heme Fe(II) complex, converted the heme Fe(III) complex into the heme Fe(III)-SH(-) complex, but the addition of Na2S did not further reduce it to the heme Fe(II) complex of the Met95Ala mutant, and no subsequent formation of the heme Fe(II)-O2 complex was observed. In contrast, a Met95His mutant formed a stable heme Fe(II)-O2 complex in response to the same treatment. An Arg97Glu mutant, containing a glutamate substitution at the amino acid that interacts with O2 in the heme Fe(II)-O2 complex, formed a stable heme Fe(II) complex in response to Na2S, but this complex failed to bind O2. Interestingly, the addition of Na2S promoted formation of verdoheme (oxygen-incorporated, modified protoporphyrin IX) in an Arg97Ile mutant. Catalytic enhancement by Na2S was similar for Met95 mutants and the wild type, but significantly lower for the Arg97 mutants. Thus, this study shows the first isolation of spectrometrically separated, stable heme Fe(III)-SH(-), heme Fe(II) and heme Fe(II)-O2 complexes of full-length EcDOS with Na2S, and confirms that external-ligand-bound, 6-coordinated heme Fe(III)-SH(-) or heme Fe(II)-O2 complexes critically contribute to the Na2S-induced catalytic enhancement of EcDOS.

Carbon monoxide binding properties of domain-swapped dimeric myoglobin.

Myoglobin (Mb) is a monomeric oxygen storage hemoprotein, and has been shown to form a domain-swapped dimer. In this study, monomeric and dimeric carbon monoxide (CO)-bound Mb (MbCO) exhibited similar absorption spectra. The CO stretching frequencies of MbCO were observed at 1,932 and 1,944 cm(-1) for both monomeric and dimeric MbCO. The resonance Raman (RR) bands for the stretching between the heme iron and axial ligands were observed at the same frequencies for the monomer and dimer of deoxygenated Mb (deoxyMb) and MbCO, respectively (ν Fe-His, 220 cm(-1); ν Fe-C, 507 cm(-1)), showing that the Fe-His bond strength of deoxyMb and the Fe-CO bond strength of MbCO did not change by the dimerization. Time-resolved RR measurements showed that the dynamics of the structural changes at the heme active site after CO photo-dissociation of MbCO was similar between monomeric and dimeric Mb [monomer, (5.2 ± 1.8) × 10(6) s(-1); dimer, (6.2 ± 1.1) × 10(6) s(-1) at room temperature]. These results show that the heme coordination structure, the protein environment around the bound CO, and the protein relaxation character are similar between monomeric and dimeric MbCO. Although the active site structure was similar between the monomer and dimer, the CO binding rate constant of dimeric Mb [(1.01 ± 0.03) × 10(6) M(-1) s(-1) at 20 °C] was about twice larger than that of the monomer [(0.52 ± 0.02) × 10(6) M(-1) s(-1) at 20 °C], presumably due to the expansion of the channel between the Xe3 cavity and the solvent by the dimerization.

Domain-swapped cytochrome cb562 dimer and its nanocage encapsulating a Zn-SO4 cluster in the internal cavity.

Protein nanostructures have been gaining in interest, along with developments in new methods for construction of novel nanostructures. We have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. Herein, we show that a four-helix bundle protein cyt cb562, with the cyt b562 heme attached to the protein moiety by two Cys residues insertion, forms a domain-swapped dimer. Dimeric cyt cb562 did not dissociate to monomers at 4 °C, whereas dimeric cyt b562 dissociated under the same conditions, showing that heme attachment to the protein moiety stabilizes the domain-swapped structure. According to X-ray crystallographic analysis of dimeric cyt cb562, the two helices in the N-terminal region of one protomer interacted with the other two helices in the C-terminal region of the other protomer, where Lys51-Asp54 served as a hinge loop. The heme coordination structure of the dimer was similar to that of the monomer. In the crystal, three domain-swapped cyt cb562 dimers formed a unique cage structure with a Zn-SO4 cluster inside the cavity. The Zn-SO4 cluster consisted of fifteen Zn2+ and seven SO42- ions, whereas six additional Zn2+ ions were detected inside the cavity. The cage structure was stabilized by coordination of the amino acid side chains of the dimers to the Zn2+ ions and connection of two four-helix bundle units through the conformation-adjustable hinge loop. These results show that domain swapping can be applied in the construction of unique protein nanostructures.

Effects of the bHLH domain on axial coordination of heme in the PAS-A domain of neuronal PAS domain protein 2 (NPAS2): Conversion from His119/Cys170 coordination to His119/His171 coordination

Neuronal PAS domain protein 2 (NPAS2), which is a CO-dependent transcription factor, consists of a basic helix–loop–helix domain (bHLH), and two heme-containing PAS domains (PAS-A and PAS-B). In our previous study on the isolated PAS-A domain, we concluded that His119 and Cys170 are the axial ligands of the ferric heme, while Cys170 is replaced by His171 upon reduction of heme (Uchida et al., J. Biol. Chem. 270, (2005) 21358–21368.). Recently, we characterized the PAS-A domain combined with the N-terminal bHLH domain, and found that some spectroscopic features were different from those of the isolated PAS-A domain (Mukaiyama et al., FEBS J. 273, (2006) 2528–2539.). Therefore, we reinvestigated the coordination structure of heme in the bHLH-PAS-A domain and prepared four histidine and one cysteine mutants. Resonance Raman spectrum of the Cys170Ala mutant is the same as that of wild type with a dominant 6-coordinate heme in the ferric form. In contrast, His119Ala and His171Ala mutants significantly increase amounts of the 5-coordinate species, indicating that His119 and His171, not Cys170, are axial ligands of the ferric heme in the bHLH-PAS-A domain. We had confirmed that the coordination structure of the isolated PAS-A domain is in equilibrium between Cys–Fe–His and His–Fe–His coordinated species but newly found that interaction of the PAS-A domain with the bHLH domain shifts the equilibrium toward the latter structure. Such flexibility in the heme coordination structure seems to be in favor of signal transduction in NPAS2.

Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH 2-truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H 2O 2 without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a five-coordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O 2) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O–O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.

Characterization of heme coordination structure in heme-DNA complex possessing gaseous molecule as an exogenous ligand

We have previously demonstrated that heme, iron(III)-protoporphyrin IX complex, and a parallel-quadruplex DNA assembled from d(TTAGGG) form a stable coordination complex called "heme-DNA complex". The heme-DNA complex exhibits a variety of spectroscopic characteristics remarkably similar to those of met-form of myoglobin, oxygen-binding hemoprotein, reflecting that the heme environments in the two systems are highly alike to each other. In a course of our effort toward exploring functional properties of the heme-DNA complex, we have investigated binding of gaseous molecules such as CO and O(2) to the heme-DNA complex. The present study revealed that the heme-DNA complex exhibits ability to accommodate these molecules as exogenous ligands.

Mechanism of redox‐ and CO‐sensing by the heme protein, CooA: Cryoradiolysis studies

Gas‐sensing heme proteins participate in a variety of signal‐transduction mechanisms across a broad range of biological systems. This class of proteins functions by undergoing an allosteric conformational change in response to the binding of a small gas molecule to a heme group. CooA, which is a redox‐ and CO‐sensing transcription factor that is found in several bacteria, regulates gene expression that enables growth on CO as a sole energy source. In the current study, we have employed the technique of gamma‐irradiation / cryoreduction spectroscopy (cryoradiolysis) to investigate facile changes that occur to the heme coordination structure of CooA during activation. Specifically, we conducted cryoradiolysis experiments to elucidate the chemical steps of an unusual ligand switch that occurs rapidly when the CooA hemes are reduced from the Fe(III) to Fe(II) state. Presented are our protocols for the preparation of samples suitable for cryoradiolysis experiments, as well as results we have obtained that provide evidence for the isolation of a highly‐reactive Fe(II)‐Cys/Pro species upon gamma irradiation at 77 K. Finally, we present initial results of annealing experiments that seek to identify the chemical details of the redox‐mediated ligand switching mechanism utilized by CooA from Rhodospirillum rubrum.The authors wish to thank Valparaiso University, the Hope College Chemistry Department, and the Riechel Fund for financial support.

Characterization of N-terminal amino group–heme ligation emerging upon guanidine hydrochloric acid induced unfolding of Hydrogenobacter thermophilus ferricytochrome c 552

Nonnative heme coordination structures emerging upon guanidine hydrochloric acid (GdnHCl) induced unfolding of Hydrogenobacter thermophilus ferricytochrome c552 were characterized by means of paramagnetic NMR. The heme coordination structure possessing the N-terminal amino group of the peptide chain in place of axial Met (His-Nterm form) was determined in the presence of GdnHCl concentrations in excess of 1.5 M at neutral pH. The stability of the His-Nterm form at pH 7.0 was found to be comparable with that of the bis-His form which has been recognized as a major nonnative heme coordination structure in cytochrome c folding/unfolding. Consequently, in addition to the bis-His form, the His-Nterm form is a substantial intermediate which affects the pathway and kinetics of the folding/unfolding of cytochromes c, of which the N-terminal amino groups are not acetylated.

Conformational Transitions of Ferricytochrome c in Strong Inorganic Acids

Conformational transitions of horse heart ferricytochromec(ferricytc) have been investigated in the presence of strong inorganic acids and their salts by optical absorption spectroscopy, magnetic circular dichroism and circular dichroism. In the presence of acids (HClO4or H2SO4, pH 2) or their salts (1 M NaClO4or Na2SO4, pH 2, 25 °C), the three ligation states of ferricytcheme were identified. One is the high-spin state: His18-Fe-H2O (40-50%), and two are the low-spin states: His18-Fe-Met80 (30-25%) and His18-Fe-His (30-25%). Under these conditions low temperatures facilitate native heme coordination of ferricytc. Transition from low-spin to high-spin heme coordination of ferricytcis complete in 1 M HClO4or 3 M H2SO4. At the concentration of HClO4and H2SO4above 3 M, different behavior in spectral transitions of ferricytcnear the heme is observed. High-spin pentacoordinated ferricytcwith the heme ligand of His18-Fe is formed in 8 M H2SO4. This state is unstable at higher concentration of H2SO4and porphyrin ferricytcis formed. At HClO4concentration higher than 3 M, the new, until this time not observed heme coordination structure of ferricytcoriginates.

3P104 鉄濃度制御蛋白質IRP2におけるヘムの配位環境(ヘム蛋白質))

3P104 鉄濃度制御蛋白質IRP2におけるヘムの配位環境(ヘム蛋白質))

Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4

A fundamental aspect of cytochrome P450 function is the role of the strictly conserved axial cysteine ligand, replacement of which by histidine has invariably resulted in mammalian and bacterial preparations devoid of heme. Isolation of the His-436 variant of NH 2-truncated P450 2B4 partly as the holoenzyme was achieved in the present study by mutagenesis of the I-helix Ala-298 residue to Glu and subsequent conversion of the axial Cys-436 to His. The expressed A298E/C436H double mutant, cloned with a hexahistidine tag, had a molecular mass equivalent to that of the primary structure of His-tagged truncated 2B4 and the sum of the two mutated residues, and contained a heme group which, when released on HPLC, showed a retention time and spectrum identical to those of iron protoporphyrin IX. The absolute spectra of A298E/C436H indicate a change in heme coordination structure from low- to high-spin, and, as expected for a His-ligated hemeprotein, the Soret maximum of the ferrous CO complex is at 422 nm. The double mutant has no oxygenase activity with representative substrates known to undergo transformation by the oxene [(FeO) 3+] or peroxo activated oxygen species, but catalyzes significant H 2O 2 formation that is NADPH- and time-dependent, and directly proportional to the concentration of A298E/C436H in the presence of saturating reductase. Moreover, the catalytic efficiency of A298E/C436H in the H 2O 2-supported peroxidation of pyrogallol is more than two orders of magnitude greater than that of wild-type 2B4 or the A298E variant. The results unambiguously demonstrate that the proximal thiolate ligand is essential for substrate oxygenation by P450.

Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c

Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82 ± 9 s −1) corresponding to the transition from the His/H 2O to the His/Met coordinated species, whereas the slow phase (24 ± 3 s −1) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.

2P063 鉄濃度制御蛋白質IRP2におけるヘムの結合とその配位環境(ヘム蛋白質)

2P063 鉄濃度制御蛋白質IRP2におけるヘムの結合とその配位環境(ヘム蛋白質)

The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase.

The extent to which the structural Ca(2+) ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca(2+) ion. The Fe(III) heme states are modified upon Ca(2+) depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca(2+)-depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca(2+) depletion are easily reversed. Contrary to the native protein, the Ca(2+)-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the nu(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca(2+) depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca(2+)-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca(2+) depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca(2+) ion as that which is lost upon Ca(2+) depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.

2P083大腸菌由来酸素センサータンパク質Dosのヘム周辺構造

2P083大腸菌由来酸素センサータンパク質Dosのヘム周辺構造

Assignment of the heme axial ligand(s) for the ferric myoglobin (H93G) and heme oxygenase (H25A) cavity mutants as oxygen donors using magnetic circular dichroism.

UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 degreesC. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known five-coordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (FeIIIPPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of FeIIIPPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.