Heme orientation modulates histidine dissociation and ligand binding kinetics in the hexacoordinated human neuroglobin

Neuroglobin (Ngb) is a globin present in the brain and retina of mammals. This hexacoordinated hemoprotein binds small diatomic molecules, albeit with lower affinity compared with other globins. Another distinctive feature of most mammalian Ngb is their ability to form an internal disulfide bridge that increases ligand affinity. As often seen for prosthetic heme b containing proteins, human Ngb exhibits heme heterogeneity with two alternative heme orientations within the heme pocket. To date, no details are available on the impact of heme orientation on the binding properties of human Ngb and its interplay with the cysteine oxidation state. In this work, we used ¹H NMR spectroscopy to probe the cyanide binding properties of different Ngb species in solution, including wild-type Ngb and the single (C120S) and triple (C46G/C55S/C120S) mutants. We demonstrate that in the disulfide-containing wild-type protein cyanide ligation is fivefold faster for one of the two heme orientations (the A isomer) compared with the other isomer, which is attributed to the lower stability of the distal His64–iron bond and reduced steric hindrance at the bottom of the cavity for heme sliding in the A conformer. We also attribute the slower cyanide reactivity in the absence of a disulfide bridge to the tighter histidine–iron bond. More generally, enhanced internal mobility in the CD loop bearing the disulfide bridge hinders access of the ligand to heme iron by stabilizing the histidine–iron bond. The functional impact of heme disorder and cysteine oxidation state on the properties of the Ngb ligand is discussed.     

Separation and identification of the regioisomers of verdoheme by reversed-phase ion-pair high-performance liquid chromatography, and characterization of their complexes with heme oxygenase

We report an HPLC method for separating the four regioisomers of verdoheme formed in the coupled oxidation of hemin with oxygen and ascorbate in aqueous pyridine. The reversed-phase ion-pair system uses hexafluoroacetone and pyridine as ion-pair agents. The regiochemistry of the separated isomers was established both by HPLC of the corresponding biliverdin IX derivatives and by 1H NMR of each isomer. Optical spectra of the pyridine verdohemochrome isomers were similar to each other, but showed differences in the absorption maxima in the red region, which appear at 680, 663, 648 and 660 nm for the alpha, beta, gamma, and delta-isomers, respectively. Each of the four isomers was incorporated anaerobically into heme oxygenase-1, yielding the corresponding verdoheme-enzyme complex. The ferrous forms had absorption maxima at 690, 667, 655, and 663 nm, and their CO-bound forms had maxima at 638, 624, 616, and 626 nm for alpha, beta, gamma, and delta-isomer, respectively. Addition of ferricyanide to the alpha-verdoheme-heme oxygenase complex brought about a ferric low-spin heme-like signal, which is identical with the ferric alpha-verdoheme complexed with the heme oxygenase that was observed in the heme oxygenase reaction.     

An N2S2 bifunctional chelator for technetium-99m and rhenium: complexation, conjugation, and epimerization to a single isomer.

A bifunctional chelator 6 was prepared bearing an N2S2 core for binding rhenium or technetium and a carboxylic acid group for conjugation to amino groups of biomolecules. Complexation of 6 with rhenium(V) resulted in two kinetic isomers, anti-7 and syn-7, being formed in approximately equal amounts. Epimerization with 0.5 M NaOH yields a single isomer anti-7, as determined by NMR spectroscopy and single-crystal X-ray analysis. The 99mTc complex was prepared at the tracer level by reaction of the ligand with 99mTcO4-, tin(II) chloride and sodium gluconate giving a mixture of two isomers, but showing a preference for the anti isomer. Chelation in the presence of 1 M NaOH results in anti-8 being formed as the sole product. The bifunctional ability of the ligand was explored by amide formation with (S)-alpha-phenethylamine, either by direct DCC coupling or through the N-hydroxy succinimidyl ester 9 intermediate. The deprotected bioconjugate 11 was complexed with rhenium, yielding similar amounts of two isomeric rhenium complexes, anti-12 and syn-12, which were isolated and characterized by NMR spectroscopy. Treatment of the kinetic mixture of anti-12 and syn-12 with 1 M NaOH resulted in quantitative conversion to a single rhenium complex anti-12. With technetium-99m in 0.1 M sodium acetate, bioconjugate 11 yielded both technetium-99m complexes anti-13 and syn-13, in a 2:1 ratio, respectively. In contrast, complexation in the presence of 1 M NaOH gave only one technetium-99m complex, assigned the structure anti-13.     

1H-NMR assignments and the dynamics of interconversion of the isomeric forms of cytochrome b5 in solution

Cytochrome b5 reconstituted with specifically deuterated hemins has led to the assignment of the resolved 6,7 beta-propionate protons and heme meso protons. Freshly reconstituted cytochrome b5 contains a mixture of two isomers in an approx. 1:1 ratio. As time proceeds the minor isomer decreases in intensity until the equilibrium ratio, approx. 8:1, of the two isomers is reached. The rate of the heme disorder kinetics was investigated for cytochrome b5 as a function of pH, oxidation state and 2,4 heme substitutents. Comparison of the kinetic data for cytochrome b5 with that obtained for other b-type heme proteins supports the proposal that the heme disorder arises from a 180 degree rotation of the heme about the alpha, gamma-meso axis. Computer-difference methods allow the spectra of the two individual isomers to be generated. Comparison of the NMR spectral parameters for the two individual isomers indicates small structural differences for amino acid side-chain orientations.     

1H NMR study of dynamics and thermodynamics of heme rotational disorder in native and reconstituted hemoglobin A

The reaction of heme and apoprotein has been studied in detail in 1H NMR spectroscopy in order to elucidate the conditions for reconstitution of hemoglobin (Hb) to yield the native protein. The initially formed holoprotein exists as a mixture of isomers with individual subunits possessing the two heme orientations differing by a 180 degrees rotation about the alpha, gamma-meso axis [La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., & Pandey, R. K. (1985) Biochemistry 24, 3826-3831]. We characterize in detail herein the rates and mechanism of heme reorientation and show that the rates differ dramatically for met-aquo and met-azido derivatives and are highly pH dependent in both subunits in a fashion that allows selective equilibration in either subunit. Nonequilibrium mixtures of such isomers can be kinetically trapped in the met-azido form and stored in this metastable form for many months. With kinetically controlled heme orientationally disordered Hb, unambiguous assignment of 1H NMR resonances to individual subunits has been made for the met-azido derivative, which demonstrates approximately 2% and 10% equilibrium heme disorder in the alpha- and beta-subunits, respectively. Comparison of the 1H NMR spectra of various heme rotationally disordered Hb derivatives indicates that this disorder is observable in all forms studied, but is most easily recognized as heme disorder and most conveniently monitored in the met-azido complex. Structural consequences of heme disorder appear to manifest themselves much more strongly in peripheral than axial interactions at the heme. Preliminary studies reveal that both the rate of autoxidation of oxy-Hb and the azide affinity of met-aquo-Hb depend on the orientation of the heme.     

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.     

Solution 1H NMR study of the active site structure for the double mutant H64Q/V68F cyanide complex from mouse neuroglobin

Solution (1)H NMR spectroscopy has been carried out to investigate the molecular and electronic structures of the active site in H64Q/V68F double mutant mouse neuroglobin in the cyanomet form. Two heme orientations resulting from a 180 degrees rotation about the alpha-gamma-meso axis were observed with a population ratio about 1:1, and the clearly distinguished B isomer was used to perform the study. Based on the analysis of the dipolar shifts and paramagnetic relaxation constants, the distal Gln(64)(E7) side chain is obtained to adopt an orientation that may produce hydrogen bond between the N(epsilon)H(1) and the Fe-bound cyanide. The side chain of Phe(68)(E11) is oriented out of the heme pocket just like that in triple mutant of cyanide complex of sperm whale myoglobin. A 15 degrees rotation of the imidazole ring in axial His(96) is observed, which is close to the varphi angle determined from the crystal structure of NgbCO. The quantitative determinations of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that cyanide is tilted by 8 degrees from the heme normal which allows for contact to the Gln(64)(E7) N(epsilon)H(1). The E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand. Furthermore, the tilt of the ligand has no obvious influence on the heme heterogeneity of cyanide ligation for isomer A/B of the wild type and mutant protein, indicating that factors other than steric effects, such as polarity of heme pocket, impacts on ligand binding affinity.     

Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.     

19F NMR of trifluoroacetyl-labeled cysteine mutants of myoglobin: structural probes of nitric oxide bound to the H93G cavity mutant.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.     

Heme oxygenase provides α-selectivity to physiological heme degradation

The isomeric composition of biliverdin formed by the degradation of heme by purified NADPH-cytochrome $\text{c}$ reductase has been determined by high performance liquid chromatography. Methemalbumin heme yields a mixture of the four biliverdin IX isomers while myoglobin yields only the IX-α isomer of biliverdin. In both cases biliverdin is a minor product of the reaction. Addition of purified heme oxygenase to the methemalbumin NADPH-cytochrome $\text{c}$ reductase system confers α-selectivity on the reaction and allows stoichiometric conversion of heme to biliverdin. Thus the role of heme oxygenase in enzymatic heme degradation appears to be to provide a suitable environment for quantitative conversion of heme to biliverdin in addition to conferring α-selectivity on the reaction.     

Structural characterization of an equilibrium unfolding intermediate in cytochrome c

Although the denaturant-induced unfolding transition of cytochrome c was initially thought to be a cooperative process, recent spectroscopic studies have shown deviations from two-state behavior consistent with accumulation of an equilibrium intermediate. However, little is known about the structural and thermodynamic properties of this state, and whether it is stabilized by the presence of non-native heme ligands. We monitored the reversible denaturant-induced unfolding equilibrium of oxidized horse cytochrome c using various spectroscopic probes, including fluorescence, near and far-UV CD, heme absorbance bands in the Soret, visible and near-IR regions of the spectrum, as well as 2D NMR. Global fitting techniques were used for a quantitative interpretation of the results in terms of a three-state model, which enabled us to determine the intrinsic spectroscopic properties of the intermediate. A well-populated intermediate was observed in equilibrium experiments at pH 5 using either guanidine-HCl or urea as a denaturant, both for wild-type cytochrome c as well as an H33N mutant chosen to prevent formation of non-native His-heme ligation. For a more detailed structural characterization of the intermediate, we used 2D 1H-15N correlation spectroscopy to follow the changes in peak intensity for individual backbone amide groups. The equilibrium state observed in our optical and NMR studies contains many native-like structural features, including a well-structured alpha-helical sub-domain, a short Trp59-heme distance and solvent-shielded heme environment, but lacks the native Met80 sulfur-iron linkage and shows major perturbations in side-chain packing and other tertiary interactions. These structural properties are reminiscent of the A-state of cytochrome c, a compact denatured form found under acidic high-salt conditions, as well as a kinetic intermediate populated at a late stage of folding. The denaturant-induced intermediate also resembles alkaline forms of cytochrome c with altered heme ligation, suggesting that disruption of the native methionine ligand favors accumulation of structurally analogous states both in the presence and absence of non-native ligands.     

1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: evidence for S = 1/2 in equilibrium S = 5/2 spin equilibrium for intact His/Met ligation

The solution 500-MHz 1H NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25 degrees C in a manner consistent with significant aggregation at low ionic strength. At high ionic strength a well-resolved 1H NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1. 2D COSY and NOESY maps at 25 degrees C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks. The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder. The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures. At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield. Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin in equilibrium high spin equilibrium with an intact His-Fe-Met coordination. The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond. The contact shifts for a coordinated Met in the high-spin state are estimated. A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations.     

Redox-related conformational changes in Rhodobacter capsulatus cytochrome c2

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.     

1H NMR studies of the effect of mutation at Valine45 on heme microenvironment of cytochrome b5

1D and 2D (1)H NMR were employed to probe the effects on the heme microenvironment of cytochrome b(5) caused by the mutation from Val45 to Tyr45, His45 and Glu45. Compared with wild type (WT) cytochrome b(5), in all mutants the heme ring are CCW rotated relative to the imidazole planes of axial ligands and the angles beta between two axial ligand imidazole planes are not changed, being in agreement with the temperature dependence of the shifts of the heme protons. The ratios of heme isomers (major to minor) are smaller than that in WT. The 4-vinyl group of the heme in V45Y assumes cis-orientation, being similar to that of WT, while in V45E and V45H, both cis and trans orientation are found. The relationships between the structure and biological function of the mutants are discussed in terms of the geometry of heme and axial ligands, the hydrophobicity of heme pocket and the electrostatic potential of the heme-exposed area.     

Metal-ion interactions with sugars. Crystal structures and FT-IR studies of the LaCl3-ribopyranose and CeCl3-ribopyranose complexes

Single crystals of LaCl3.C5H10O5.5H2O (1) and CeCl3.C5H10O5.5H2O (2) were obtained from ethanol-water solutions and their structures determined by X-ray. The two complexes are isomorphous. Two configurations of complex 1 or complex 2, as a pair of isomers, were found in each single crystal in a disordered state. The ligand of one of the isomer is alpha-D-ribopyranose in the 4C1 conformation, the ligand of the other is beta-D-ribopyranose in the 1C4 conformation. For complex 1, the alpha:beta anomeric ratio is 51:49, and for complex 2, the ratio is 52:48. Both ligands of the two isomers provide three hydroxyl groups in ax-eq-ax orientation for coordination. The Ln3+ (Ln = La or Ce) ion is nine-coordinated with five Ln-O bonds from water molecules, three Ln-O bonds from hydroxyl groups of the D-ribopyranose, and one Ln-Cl bond from chloride ion. The hydroxyl groups, water molecules, and chloride ions form an extensive hydrogen-bond network. The IR spectral C-C, O-H, C-O, and C-O-H vibrations were observed to be shifted in both the two complexes and the IR results are in accord with those of X-ray diffraction.     

Chemistry of sulforhodamine--amine conjugates

Commercially-available sulforhodamine sulfonyl chlorides contain two isomeric monosulfonyl chlorides. Conjugates of these isomers with amines have different properties because the sulfonamide formed from one isomer can undergo ring-closure to a colorless sultam. This chemistry has been examined for a model conjugate with methylamine and for a bioconjugate with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP. The interaction of each isomer of the latter conjugates with myosin subfragment 1 has been characterized. Significant differences between the two isomers are observed in these interactions.     

Structural insights into the inclusion complexes between clomiphene citrate and β‐cyclodextrin: The mechanism of preferential isomeric selection

A major challenge in pharmaceuticals for clinical applications is to alter the solubility, stability, and toxicity of drug molecules in living systems. Cyclodextrins (CDs) have the ability to form host–guest inclusion complexes with pharmaceuticals for further development of new drug formulations. The inclusion complex of clomiphene citrate (CL), a poorly water‐soluble drug, with native β‐cyclodextrin (β‐CD) was characterized by a one and two‐dimensional nuclear magnetic resonance (NMR) spectroscopic approach and also by molecular docking techniques. Here we report NMR and a computational approach in preferential isomeric selection of CL, which exists in two stereochemical isomers, enclomiphene citrate (ENC; E isomer) and zuclomiphene citrate (ZNC; Z isomer) with β‐CD. β‐CD cavity protons, namely, H‐3′ and H‐5′, experienced shielding in the presence of CL. The aromatic ring protons of the CL molecule were observed to be deshielded in the presence of β‐CD. The stoichiometric ratio of the β‐CD:CL inclusion complex was observed by NMR and found to be 1:1. The overall binding constant of β‐CD:CL inclusion complexes was based on NMR chemical shifts and was calculated to be 50.21 M⁻¹. The change in Gibb's free energy (∆G) was calculated to be −9.80 KJ mol⁻¹. The orientation and structure of the β‐CD:CL inclusion complexes are proposed on the basis of NMR and molecular docking studies. 2D ¹H‐¹H ROESY confirmed the involvement of all three aromatic rings of CL in the inclusion complexation with β‐CD in the solution, confirming the multiple equilibria between β‐CD and CL. Molecular docking and 2D ¹H‐¹H ROESY provide insight into the inclusion complexation of two isomers of CL into the β‐CD cavity. A molecular docking technique further provided the different binding affinities of the E and Z isomers of CL with β‐CD and confirmed the preference of the Z isomer binding for β‐CD:CL inclusion complexes. The study indicates that the formation of a hydrogen bond between –O– of CL and the hydrogen atom of the hydroxyl group of β‐CD was the main factor for noncovalent β‐CD:CL inclusion complex formation and stabilization in the aqueous phase.     

Purification of synthetic all-E lycophyll (psi,psi-carotene-16,16'-diol)

An efficient purification of synthetic all-trans (all-E) lycophyll is described. The synthetic preparation of the rare xanthophyll lycophyll produces a mixture of geometric isomers. Purification by HPLC using reverse-phase C30 silica affords milligram quantities of the desired all-trans isomer in >95% purity, as confirmed by (1)H NMR and LC/MS. Most recently, a facile work-up of the geometric mixture formed during total synthesis was found to provide multigrams of the targeted all-E geometric isomer of lycophyll. The acquisition of modest quantities of this specific lycopene analog allows its therapeutic potential to be explored.     

Cytochrome c551 from Starkeya novella: characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulfidophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: +133 mV (pH 6.0); +104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxX, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.     

Mesopone cytochrome c peroxidase: functional model of heme oxygenated oxidases

The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin derivatives were formed by OsO(4) treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement. The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl(2) and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP. The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c. EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp(191) radical similar to wild-type CcP in the reaction cycle. Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is approximately 85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a approximately 60-mV increase in Fe(III)/Fe(II) reduction potential. The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 A. Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein.