Biomedical subjects

C Hemann

Publications and source records attributed to C Hemann.

8 recordsLinked to original sources

PubMed2001

A conserved tryptophan in nitric oxide synthase regulates heme-dioxy reduction by tetrahydrobiopterin.

In nitric oxide synthase (NOS), (6R)-tetrahydrobiopterin (H(4)B) binds near the heme and can reduce a heme-dioxygen intermediate (Fe(II)O(2)) during Arg hydroxylation [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. A conserved Trp engages in aromatic stacking with H(4)B, and its mutation inhibits NO synthesis. To examine how this W457 impacts H(4)B redox function, we performed single turnover reactions with the mouse inducible NOS oxygenase domain (iNOSoxy) mutants W457F and W457A. Ferrous mutants containing Arg and H(4)B were mixed with O(2)-containing buffer, and then heme spectral transitions, H(4)B radical formation, and Arg hydroxylation were followed versus time. A heme Fe(II)O(2) intermediate was observed in W457A and W457F and had normal spectral characteristics. However, its disappearance rate (6.5 s(-1) in W457F and 3.0 s(-1) in W457A) was slower than in wild-type (12.5 s(-1)). Rates of H(4)B radical formation (7.1 s(-1) in W457F and 2.7 s(-1) in W457A) matched their rates of Fe(II)O(2) disappearance, but were slower than radical formation in wild-type (13 s(-1)). The extent of H(4)B radical formation in the mutants was similar to wild-type, but their radical decayed 2-4 times faster. These kinetic changes correlated with slower and less extensive Arg hydroxylation by the mutants (wild-type > W457F > W457A). We conclude that W457 ensures a correct tempo of electron transfer from H(4)B to heme Fe(II)O(2), possibly by stabilizing the H(4)B radical. Proper control of these parameters may help maximize Arg hydroxylation and minimize uncoupled O(2) activation at the heme.

Animals↗

PubMed2001

Protein-coenzyme interactions in adenosylcobalamin-dependent glutamate mutase.

Glutamate mutase catalyses an unusual isomerization involving free-radical intermediates that are generated by homolysis of the cobalt-carbon bond of the coenzyme adenosylcobalamin (coenzyme B(12)). A variety of techniques have been used to examine the interaction between the protein and adenosylcobalamin, and between the protein and the products of coenzyme homolysis, cob(II)alamin and 5'-deoxyadenosine. These include equilibrium gel filtration, isothermal titration calorimetry, and resonance Raman, UV-visible and EPR spectroscopies. The thermodynamics of adenosylcobalamin binding to the protein have been examined and appear to be entirely entropy-driven, with DeltaS=109 J.mol(-1).K(-1). The cobalt-carbon bond stretching frequency is unchanged upon coenzyme binding to the protein, arguing against a ground-state destabilization of the cobalt-carbon bond of adenosylcobalamin by the protein. However, reconstitution of the enzyme with cob(II)alamin and 5'-deoxyadenosine, the two stable intermediates formed subsequent to homolysis, results in the blue-shifting of two of the bands comprising the UV-visible spectrum of the corrin ring. The most plausible interpretation of this result is that an interaction between the protein, 5'-deoxyadenosine and cob(II)alamin introduces a distortion into the ring corrin that perturbs its electronic properties.

Cobamides↗

PubMed2001

Rapid kinetic studies link tetrahydrobiopterin radical formation to heme-dioxy reduction and arginine hydroxylation in inducible nitric-oxide synthase.

To understand how heme and (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B) participate in nitric-oxide synthesis, we followed ferrous-dioxy heme (Fe(II)O(2)) formation and disappearance, H(4)B radical formation, and Arg hydroxylation during a single catalytic turnover by the inducible nitric-oxide synthase oxygenase domain (iNOSoxy). In all cases, prereduced (ferrous) enzyme was rapidly mixed with an O(2)-containing buffer to start the reaction. A ferrous-dioxy intermediate formed quickly (53 s(-1)) and then decayed with concurrent buildup of ferric iNOSoxy. The buildup of the ferrous-dioxy intermediate preceded both H(4)B radical formation and Arg hydroxylation. However, the rate of ferrous-dioxy decay (12 s(-1)) was equivalent to the rate of H(4)B radical formation (11 s(-1)) and the rate of Arg hydroxylation (9 s(-1)). Practically all bound H(4)B was oxidized to a radical during the reaction and was associated with hydroxylation of 0.6 mol of Arg/mol of heme. In dihydrobiopterin-containing iNOSoxy, ferrous-dioxy decay was much slower and was not associated with Arg hydroxylation. These results establish kinetic and quantitative links among ferrous-dioxy disappearance, H(4)B oxidation, and Arg hydroxylation and suggest a mechanism whereby H(4)B transfers an electron to the ferrous-dioxy intermediate to enable the formation of a heme-based oxidant that rapidly hydroxylates Arg.

Animals↗

PubMed1999

Rubredoxin from the green sulfur bacterium Chlorobium tepidum functions as an electron acceptor for pyruvate ferredoxin oxidoreductase.

Rubredoxin (Rd) from the moderately thermophilic green sulfur bacterium Chlorobium tepidum was found to function as an electron acceptor for pyruvate ferredoxin oxidoreductase (PFOR). This enzyme, which catalyzes the conversion of pyruvate to acetyl-CoA and CO(2), exhibited an absolute dependence upon the presence of Rd. However, Rd was incapable of participating in the pyruvate synthase or CO(2) fixation reaction of C. tepidum PFOR, for which two different reduced ferredoxins are employed as electron donors. These results suggest a specific functional role for Rd in pyruvate oxidation and provide the initial indication that the two important physiological reactions catalyzed by PFOR/pyruvate synthase are dependent on different electron carriers in the cell. The UV-visible spectrum of oxidized Rd, with a monomer molecular weight of 6500, gave a molar absorption coefficient at 492 nm of 6.89 mM(-1) cm(-1) with an A(492)/A(280) ratio of 0.343 and contained one iron atom/molecule. Further spectroscopic studies indicated that the CD spectrum of oxidized C. tepidum Rd exhibited a unique absorption maximum at 385 nm and a shoulder at 420 nm. The EPR spectrum of oxidized Rd also exhibited unusual anisotropic resonances at g = 9.675 and g = 4.322, which is composed of a narrow central feature with broader shoulders to high and low field. The midpoint reduction potential of C. tepidum Rd was determined to be -87 mV, which is the most electronegative value reported for Rd from any source.

Chlorobi↗

PubMed1998

Formation of a tyrosyl radical in xanthine oxidase.

Treatment of xanthine oxidase with ferricenium at high pH gives rise to an EPR signal not previously seen with this enzyme. The signal is apparently isotropic at 9 GHz with a gavg of approximately 2 and once generated is stable to pH 6.0, so long as the sample is kept in the dark. Treatment of the signal-giving species with hydroxyurea results in complete loss of the signal, indicating that the signal is radical-based. Pretreatment of the enzyme with iodoacetate has no effect on signal formation with ferricenium. The ferricenium-generated EPR signal shows proton hyperfine coupling that is not lost upon exchange into D2O and bears considerable resemblance to the tyrosyl radical of the photosynthetic reaction center and other systems. These observations lead us to interpret the new ferricenium-generated EPR signal of xanthine oxidase as arising from a tyrosyl radical, the result of one-electron oxidation of a protein tyrosinate residue. Kinetic parameters for the reductive half-reaction of ferricenium-treated xanthine oxidase with xanthine were determined by stopped-flow spectrophotometry; kred and KDxanthine (15 s-1 and 12 microM, respectively) were essentially unchanged. Addition of 2-hydroxy-6-methylpurine (in the presence of 2 mg/mL catalase and superoxide dismutase) generated the "very rapid" MoV EPR signal while preserving the ferricenium-derived EPR signal, providing a further indication that the modified enzyme remains fully functional and the presence of the tyrosyl radical does not impact turnover by the enzyme. Coupling of the two signals was not evident, nor was coupling to the two 2Fe-2S centers or the flavin semiquinone evident. The implications of covalent modifications of proteins mediated by ferricenium are discussed.

Animals↗

PubMed1998

FTIR characterization of heterocycles lumazine and violapterin in solution: effects of solvent on anionic forms.

Fourier transform infrared (FTIR) spectra have been obtained from solution samples of the heterocycles uracil, lumazine, and violapterin and reveal interpretable carbonyl stretching frequencies. Spectra of conjugate bases of lumazine and violapterin demonstrate decreases in these carbonyl stretching frequencies upon ionization. Based on isotopic shifts from amide deuterated analogs, semiempirical QCFF/PI calculations were used to assign the vibrational frequencies in the region 1100-1800 cm-1 observed from samples in dimethylsulfoxide (DMSO) and aqueous solutions to specific normal modes. The observed deuterium shifts and the calculations suggest that, in some cases, N-H bending motions are coupled to the C=O stretching motions of the pyrimidine ring. These data suggest that for lumazine anions a change in solvent can significantly change the mixing of the N-H bending and C=O stretching vibrational motions. This implies that vibrational analysis for lumazine species in relatively noninteracting media like nonpolar solvents, mulls or pellets cannot necessarily be transferred to the system when it is dissolved in a polar, hydrogen-bonding solvent such as water. Although other explanations can be offered, our vibrational analysis suggests that the changes in normal mode composition of the predominantly C=O stretching vibrations of lumazine anion on going from dimethylsulfoxide to water solution are consistent with a change in the predominant tautomer of the heterocycle. This change appears to correspond to a shifting of the location of the remaining acidic proton to a different ring nitrogen atom. This interpretation is of interest in view of recent ab initio calculations which suggest that proton shifts may occur during the hydroxylation of lumazine as mediated by the enzyme xanthine oxidase.

Anions↗

PubMed1994

High-copy expression vector based on amplification-promoting sequences.

We describe a new vector system that allows efficient expression of heterologous proteins in transformed mouse L fibroblasts. This is due to its persistence at high copy numbers, achieved by a 370-bp amplification promoting element (muNTS1) derived from the nontranscribed spacer of murine rDNA. Copy number determination showed that this sequence mediates a 40- to 800-fold amplification of the vector DNA in transfected L cells. High copy number was accompanied by increased expression levels of the reporter gene secreted alkaline phosphatase (SEAP). Analyzing the structural organization of multicopy plasmid DNA in mouse L cells revealed that plasmid DNA is integrated as reiterated head-to-tail concatamers into the chromosomal DNA. The vector described here can be used as a versatile high-copy expression system for heterologous proteins overcoming any limitation to enzyme-deficient cell lines.

Alkaline Phosphatase↗

PubMed1993

Reductive half-reaction of xanthine oxidase: mechanistic role of the species giving rise to the "rapid type 1" molybdenum(V) electron paramagnetic resonance signal.

The reaction of xanthine oxidase with xanthine, 1-methylxanthine, and 2-hydroxy-6-methylpurine has been reinvestigated with the aim of elucidating the mechanistic role of the species giving rise to the "rapid" Mo(V) electron paramagnetic resonance (EPR) signal. It is found that addition of 2.0 mM 1-methylxanthine or 2-hydroxy-6-methylpurine to partially reduced enzyme generates substantial amounts of the Type 1 form of the "rapid" EPR signal, characterized by superhyperfine coupling to one strongly interacting (aav = 13 G) and one weakly interacting (aav = 3 G) proton. The "rapid" signals observed with both substrates are identical to those observed in the course of the anaerobic reaction of enzyme with a stoichiometric excess of substrate. With 2-hydroxy-6-methylpurine at pH 10, a burst phase in the formation of the species giving rise to the "rapid Type 1" signal is observed that is fast relative to the rate of formation of the species giving rise to the "very rapid" EPR signal. At pH 8.5, partial reduction of enzyme prior to reaction with xanthine, 1-methylxanthine, or 2-hydroxy-6-methylpurine reverses the relative amounts of "rapid" and "very rapid" EPR signal observed at the shortest reaction times. The substantial amounts of "rapid Type 1" signal formed by addition of substrates to partially reduced enzyme or by reaction of oxidized enzyme with a stoichiometric excess of substrate contrasts with previous work, which has shown that under single-turnover conditions none of the substrates investigated generates an appreciable amount of "rapid" EPR signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗