Papers by Mario Alberto Acosta Rivera
Journal of the American Chemical Society, 2003
Mutation of His-39, one of the axial ligands in rat outer mitochondrial membrane cytochrome b5 (O... more Mutation of His-39, one of the axial ligands in rat outer mitochondrial membrane cytochrome b5 (OM cyt b5), to Val produces a mutant (H39V) capable of carrying out the oxidation of heme to biliverdin when incubated with hydrazine and O2. The reaction proceeds via the formation of an oxyferrous complex (Fe II-O2) that is reduced by hydrazine to a ferric hydroperoxide (Fe III-OOH) species. The latter adds a hydroxyl group to the porphyrin to form meso-hydroxyheme. The observation that catalase does not inhibit the oxidation of the heme in the H39V mutant is consistent with the formation of a coordinated hydroperoxide (Fe III-OOH), which in heme oxygenase is the precursor of meso-hydroxyheme. By comparison, mutation of His-63, the other axial ligand in OM cyt b5, to Val results in a mutant (H63V) capable of oxidizing heme to verdoheme in the absence of catalase. However, the oxidation of heme by H63V is completely inhibited by catalase. Furthermore, whereas the incubation of Fe III-H63V with H2O2 leads to the nonspecific degradation of heme, the incubation of Fe II-H63V with H2O2 results in the formation of meso-hydroxyheme, which upon exposure to O2 is rapidly converted to verdoheme. These findings revealed that although mesohydroxyheme is formed during the degradation of heme by the enzyme heme oxygenase or by the process of coupled oxidation of model hemes and hemoproteins not involved in heme catabolism, the corresponding mechanisms by which meso-hydroxyheme is generated are different. In the coupled oxidation process O2 is reduced to noncoordinated H2O2, which reacts with Fe II-heme to form meso-hydroxyheme. In the heme oxygenation reaction a coordinated O2 molecule (Fe II-O2) is reduced to a coordinated peroxide molecule (Fe III-OOH), which oxidizes heme to meso-hydroxyheme.
Journal of the American Chemical Society, 2005
Journal of the American Chemical Society, 2005
The azide complexes of heme oxygenase from Pseudomonas aeruginosa (pa-HO) and Neisseriae meningit... more The azide complexes of heme oxygenase from Pseudomonas aeruginosa (pa-HO) and Neisseriae meningitidis (nm-HO) have been studied with the aid of 1 H and 13 C NMR spectroscopy. These complexes have been shown to exist as an equilibrium mixture of two populations, one exhibiting an S) 1 /2, (dxy) 2 (dxz, dyz) 3 electron configuration and planar heme and a second with a novel S) 3 /2, (dxz, dyz) 3 (dxy) 1 (dz 2) 1 spin state and nonplanar heme. At physiologically relevant temperatures, the equilibrium shifts in the direction of the population exhibiting the latter electron configuration and nonplanar heme, whereas at temperatures approaching the freezing point of water, the equilibrium shifts in the direction of the population with the former electronic structure and planar heme. These findings indicate that the microenvironment of the distal pocket in heme oxygenase is unique among heme-containing proteins in that it lowers the σ-donating (field strength) ability of the distal ligand and, therefore, promotes the attainment of heme electronic structures thus far only observed in heme oxygenase. When the field strength of the distal ligand is slightly lower than that of azide, such as OH-(J. Am. Chem. Soc. 2003, 125, 11842), the corresponding complex exists as a mixture of populations with nonplanar hemes and electronic structures that place significant spin density at the meso positions. The ease with which these unusual heme electronic structures are attained by heme oxygenase is likely related to activation of meso carbon reactivity which, in turn, facilitates hydroxylation of a meso carbon by the obligatory ferric hydroperoxide intermediate.
Journal of the American Chemical Society, 2003
13 C NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeru... more 13 C NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase (Fe III-OH), where OHhas been used as a model of the OOHligand to gain insights regarding the elusive ferric hydroperoxide (Fe III-OOH) intermediate in heme catabolism at ambient temperatures. Analysis of the heme core carbon resonances revealed that the coordination of hydroxide in the distal site of the enzyme results in the formation of at least three populations of Fe III-OH complexes with distinct electronic configurations and nonplanar ring distortions that are in slow exchange relative to the NMR time scale. The most abundant population exhibits a spin crossover between S) 1 /2 and S) 3 /2 spin states, and the two less abundant populations exhibit pure, S) 3 /2 and S) 1 /2, (dxy) 1 electronic configurations. We propose that the highly organized network of water molecules in the distal pocket of heme oxygenase, by virtue of donating a hydrogen bond to the coordinated hydroxide ligand, lowers its ligand field strength, thereby increasing the field strength of the porphyrin (equatorial) ligand, which results in nonplanar deformations of the macrocycle. This tendency to deform from planarity, which is imparted by the ligand field strength of the coordinated OH-, is likely reinforced by the flexibility of the distal pocket in HO. These findings suggest that if the ligand field strength of the coordinated OOHin heme oxygenase is modulated in a similar manner, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.
Journal of the American Chemical Society, 2002
The origin of the unusual regioselectivity of heme oxygenation, ie the oxidation of heme to δ-bil... more The origin of the unusual regioselectivity of heme oxygenation, ie the oxidation of heme to δ-biliverdin (70%) and β-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by 1 H NMR, 13 C NMR, and resonance Raman ...
Journal of Magnetic Resonance, 1998
In paramagnetic heme proteins, it is often problematic to make the reduced (usually diamagnetic) ... more In paramagnetic heme proteins, it is often problematic to make the reduced (usually diamagnetic) state lack isotropic shifts proton resonance assignments for heme substituents that do not and are therefore difficult to examine by NMR spectroscopy. have large isotropic shifts and consequently lie under the large Observation of these resonances is important in order to envelope of polypeptide resonances. Furthermore, assignments study structure-function relationships in electron-transfer that would normally be performed with the aid of HMBC experiheme proteins, which function by shuttling their heme iron ments in diamagnetic molecules can prove difficult in the active between ferric and ferrous oxidation states. (c) Efficient site of paramagnetic heme proteins if T 01 2 ú 2 J CH. To circumvent spin-spin relaxation often makes through-bond proton-prothis problem, a new method is presented to selectively detect 1 H ton correlations in COSY or TOCSY experiments unobservin 1 H n-13 C-13 C fragments biosynthetically introduced into the able (T 01 2 ú J HH) (2, 6). In fact, it has been proposed that active site of heme proteins. The pulse sequence combines wellthe COSY cross peaks observed in paramagnetic systems known building blocks such as INEPT to transfer 1 H spin magnetization to bonded 13 C nuclei, followed by INADEQUATE to gener-arise from dipolar coupling and Curie spin-nuclear spin ate 13 C-13 C double-quantum coherence that is selected with relaxation (7, 8). pulsed field gradients, and finally reverse-INEPT to transfer mag-One strategy designed to circumvent some of these probnetization back to 1 H nuclei for subsequent observation. The new lems involves the selective 13 C enrichment of heme substitu-1 H n-13 C-13 C edited experiment takes advantage of the relatively ents and their subsequent observation by 13 C NMR spectroslarge values of 1 J CH and 1 J CC , avoiding the long interpulse delays copy. We have recently reported a biosynthetic method for in HMBC that compromise the detectability of rapidly relaxing the expression of 13 C-labeled heme cytochrome b 5 (9), thus nuclei. The potential applicability of the pulse sequence is demonfacilitating NMR experiments such as HMQC and HSQC strated by its contribution to the unambiguous assignment of the (10-14) that rely on the heteronuclear through-bond concarbonyl carbons in the heme propionates of ferricytochrome b 5. nectivity and larger scalar coupling constant 1 J CH. However,
Journal of Labelled Compounds and Radiopharmaceuticals, 1997
Journal of Inorganic Biochemistry, 2005
The activation of dioxygen by heme oxygenase proceeds via formation of an obligatory ferric hydro... more The activation of dioxygen by heme oxygenase proceeds via formation of an obligatory ferric hydroperoxide intermediate (FeIII-OOH), as is the case in the activation of dioxygen by monooxygenase enzymes. This review summarizes current understanding of the structural and dynamic properties in heme oxygenase that channel the reactivity of the FeIII-OOH intermediate toward heme hydroxylation rather than oxoferryl formation. In addition, structural and electronic factors dictating the regiospecificity of heme oxygenation are analyzed in the context of recent X-ray and NMR spectroscopic studies. Differences in mechanism between heme hydroxylation, as carried out by heme oxygenase, and the coupled oxidation process, are also addressed.
JBIC Journal of Biological Inorganic Chemistry, 1999
Singly and doubly labeled delta-aminolevulinic acid derivatives were used to prepare rat liver ou... more Singly and doubly labeled delta-aminolevulinic acid derivatives were used to prepare rat liver outer mitochondrial membrane (OM) cytochrome b5 containing a 13C-labeled heme active site. A variety of NMR experiments, including HMBC and INADEQUATE in conjunction with the more commonly used HMQC, NOESY, and COSY, were conducted to make unambiguous assignments of protonated carbons and the quaternary pyrrole-alpha and -beta carbons in both isomeric forms of the paramagnetic active center of OM cytochrome b5. Because the long interpulse delays in the HMBC experiment have a detrimental effect on the detectability of fast relaxing paramagnetically affected resonances. INADEQUATE is proposed as the experiment of choice for assigning quaternary carbons in paramagnetic hemes with carefully chosen macrocycle labeling patterns. Furthermore, the applicability of the INADEQUATE experiment to paramagnetic heme active sites should be facilitated greatly by the availability of biosynthetic methods for producing isotopically labeled b-hemes and, more recently, isotopically labeled c-hemes.
Journal of Biological Chemistry, 2004
Inorganic Chemistry, 2006
Evidence is presented demonstrating that the magnitudes of the 13 C chemical shifts originating f... more Evidence is presented demonstrating that the magnitudes of the 13 C chemical shifts originating from heme meso carbons provide a straightforward diagnostic tool to elucidate the coordination state of high-spin heme proteins and enzymes. Pentacoordinate high-spin heme centers exhibit 13 C meso shifts centered at approximately 250 ppm, whereas their hexacoordinate counterparts exhibit 13 C shifts centered at approximately −80 ppm. The relatively small spectral window (400 to −100 ppm) covering the meso-13 C shifts, the relatively narrow lines of these resonances, and the availability of biosynthetic methods to prepare 13 C-labeled heme (Rivera, M.; Walker, F. A. Anal. Biochem. 1995, 230, 295−302) make this approach practical. The theoretical basis for the distinct chemical shifts observed for meso carbons from hexacoordinate high-spin hemes relative to their pentacoordinate counterparts are now well understood (Cheng, R.-J.; Chen, P. Y.; Lovell, T.; Liu, T.; Noodleman, L.; Case, D. A. J. Am. Chem. Soc. 2003, 125, 6774−6783), which indicates that the magnitude of the meso-carbon chemical shifts can be used as a simple and reliable diagnostic tool for determining the coordination state of the heme active sites, independent of the nature of the proximal ligand. Proof of the principle for the 13 C NMR spectroscopic approach is demonstrated using hexa-and pentacoordinate myoglobin. Subsequently, 13 C NMR spectroscopy has been used to unambiguously determine that a recently discovered heme protein from Shigella dysenteriae (ShuT) is pentacoordinate.
Inorganic Chemistry, 2004
The ferrous R-verdoheme−cytochrome b 5 complex, [Fe II (verdoheme)] + , has been prepared and cha... more The ferrous R-verdoheme−cytochrome b 5 complex, [Fe II (verdoheme)] + , has been prepared and characterized spectroscopically. Anaerobic addition of excess sodium dithionite to [Fe II (verdoheme)] + at pH 10 produces a oneelectron-reduced species with spectroscopic characteristics that suggest a ferrous hexacoordinated verdoheme π neutral radical best formulated as a [Fe II (verdoheme•)] f [Fe I (verdoheme)] resonance hybrid. At lower pH values (7.0 and 8.0) the one-electron-reduced species is shown to disproportionate to produce the resting state [Fe II-(verdoheme)] + complex and the two-electron-reduced [Fe II (verdoheme:)]anion. The latter might also be formulated as a resonance hybrid [Fe I (verdoheme•)]f [Fe II (verdoheme:)]-. The disproportionation reaction becomes very slow as the pH is raised above 9.0. Exposure of the one-electron-or two-electron-reduced verdoheme complexes of cytochrome b 5 to O 2 results in rapid and quantitative reoxidation to the resting state [Fe II (verdoheme)] + complex.
Biochemistry, 2005
Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeru... more Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IXdelta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.
Biochemistry, 2004
It has been reported that the R183E and R183D mutants of rat heme oxygenase-1 (r-HO-1) produce ap... more It has been reported that the R183E and R183D mutants of rat heme oxygenase-1 (r-HO-1) produce approximately 30% delta-biliverdin [Zhou, H., et al. (2000) J. Am. Chem. Soc. 122, 8311-8312]. Two plausible mechanisms were proposed to explain the observations. (a) Electrostatic repulsion between E183 (D183) and one of the heme propionates forces the heme to rotate, thereby placing the delta-meso carbon in a position that is susceptible to oxidation. (b) Rearrangement of the distal pocket structure is triggered by the formation of a hydrogen bond between E183 (D183) and K179. A change in the pK(a) for the Fe(III)-H(2)O to Fe(III)-OH transition of the mutants was interpreted to be consistent with rearrangement of the hydrogen bond network in the distal pocket. The large similarities between the high-frequency portion of the (1)H NMR spectra corresponding to the wild type and R183E and R183D mutants were interpreted to indicate that the heme in the mutants is not rotated to a significant extent. We have re-examined this issue by studying the corresponding R177 mutants in heme oxygenase from Corynebacterium diphtheriae (cd-HO). Replacing R177 with E or D results in the formation of approximately 55% alpha- and 45% delta-biliverdin, whereas the R177A mutant retains alpha-regioselectivity. In addition, the K13N/Y130F/R177A triple mutant catalyzed the formation of 60% delta- and 40% alpha-biliverdin, while single mutants K13N and Y130F did not appreciably change the regioselectivity of the reaction. The pK(a) of the Fe(III)-H(2)O to Fe(III)-OH transition in wild-type cd-HO is 9.1, and those of the R177E, R177D, R177A, and K13N/Y130F/R177A mutants are 9.4, 9.5, 9.2, and 8.0, respectively. Thus, no obvious correlation exists between the changes in pK(a) and the altered regioselectivity. NMR spectroscopic studies conducted with the R177D and R177E mutants of cd-HO revealed the presence of three heme isomers: a major (M) and a minor (m) heme orientational isomer related by a 180 degrees rotation about the alpha-gamma meso axis and an alternative seating (m') which is related to m by an 85 degrees in-plane rotation of the macrocycle. The in-plane rotation of m to acquire conformation m' is triggered by electrostatic repulsion between the side chains of D or E at position 177 and heme propionate-6. As a consequence, the delta-meso carbon in m' is placed in the position occupied by the alpha-meso carbon in m, where it is susceptible to hydroxylation and subsequent formation of delta-biliverdin.
Biochemistry, 2009
The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in E. coli. The resultant prot... more The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in E. coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex0020from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5-10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 ± 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH the heme in BfrB remains oxidized and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR and the apo-form of P. aeruginosa bacterioferritin associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [
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Papers by Mario Alberto Acosta Rivera