Human Heme Oxygenase Oxidation of 5- and 15-Phenylhemes

Human heme oxygenase-1 (hHO-1) catalyzes the O2- dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the -meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IX . Surprisingly, a 15-methyl substituent caused exclusive cleavage at the -meso- rather than at the normal, unsubstituted -meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IX and oxidizes 15- phenylheme at the -meso position to give 10-phenylbiliverdin IX . The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-Å crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141–150 and in the proximal Lys18 and Lys22. In the 5-phenylhemehHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26–42 near the -meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.Fil: Wang, Jingling. University of California; Estados UnidosFil: Niemevz, Fernando. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Lad, Latesh. University of California; Estados UnidosFil: Huang, Liusheng. University of California; Estados UnidosFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Buldain, Graciela Yolanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Poulos, Thomas L.. University of California; Estados UnidosFil: Ortiz de Montellano, Paul R.. University of California; Estados Unido

The catalytic mechanism of ascorbate peroxidase : probing the effects of changes in enzyme and substrate structure

Recombinant pea cytosolic ascorbate peroxidase (rAPX) has been isolated and the mechanistic properties have been investigated. Rate constants for formation of compound I with H2O2 and other organic peroxides were measured. The data indicate that the structure and size of peroxide dictate the rate of compound I formation. Rate constants for reduction of compound I and compound II were measured with L-ascorbic acid and other derivatised forms of ascorbate. Reduction of compound II showed evidence for formation of an enzyme-substrate complex. The rate constants for compound II reduction, by the various ascorbate-based substrates, were controlled by the thermodynamic driving force of the reaction.;Variants H42A and H42E were constructed to investigate the catalytic role of His42 in rAPX catalysis. The observed pseudo-first-order rate constant for the reaction between the His42 variants and hydrogen peroxide saturates at high peroxide concentration. The data are consistent with a two-step mechanism involving the formation of an APX-H2O2 intermediate whose conversion to compound I is rate-limiting. pH-Dependence studies on compound I formation reveal His42 as the key ionizable residue. Rapid photodiode array spectrophotometry revealed the presence of a transient intermediate for H42A, with a spectrum consistent with a ferric-hydroperoxy complex. The rate of formation of compound I and peroxidase activity of H42E were significantly greater than H42A, however, addition of exogenous imidazoles partially rescues both the rate of compound I formation and peroxidase activity for H42A

Lipid sensing apolipoprotein A-I for novel high -throughput lipidation assays

Apolipoprotein A-I (ApoA-I) is the primary protein component of high density lipoproteins (HDL). ApoA-I plays an important role in cholesterol metabolism by mediating the formation of nascent HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by the ATP-binding cassette A1 (ABCA1). Insufficient ABCA1 activity my lead to reduced HDL formation, reduced cholesterol efflux and the development of arteriosclerosis. The standard radioactive assay for measuring cholesterol transport to lipid-free ApoA-I has low through-put, poor dynamic range and fails to measure phospholipid transferred with cholesterol. We describe the development of two sensitive, non-radioactive high-throughput assays that report on the lipidation state of ApoA-I and may have applications for studying ABCA1 function and HDL metabolism: a homogenous assay based on the Time Resolved FRET (TR-FRET) and a discontinuous assay that uses the Epic. The TR-FRET assay employs a fluorescent ApoA-I where Cysteine is labeled with the FRET acceptor HiLyte-Fluor-647 and an N-terminal Biotin-AviTag is bound to the streptavidin-Terbium conjugate. When this ApoA-I was incorporated into recombinant HDL, TR-FRET decreased proportionally to the increase in the ratio of lipid to ApoA-I in agreement with the expansion of the surface area of lipids concomitant with the increase in separation of N-terminal and central regions of the protein and demonstrated that the HTRF assay was sensitive to the amount of lipid associated with ApoA-I. The Epic is a label-free platform that allows for the observation of direct biomolecular interactions via a resonant wavelength shift which is proportional to the mass bound to the surface. In the Epic assay, biotinylated ApoA-I was captured on streptavidin-coated sensor. The response was proportional to the amount of lipids associated with ApoA-I indicating that the assay could sense lipidation of ApoA-I.Peer reviewed: YesNRC publication: Ye