Xuhong Zhang

Heme degradation as catalyzed by a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.

Hmu O, a heme degradation enzyme in the pathogenCorynebacterium diphtheriae, catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. A bacterial expression system using a synthetic gene coding for the 215-amino acid, full-length Hmu O has been constructed. Expressed at very high levels in Escherichia coli BL21, the enzyme binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converts hemin to biliverdin with α-hydroxyhemin and verdoheme as intermediates. The overall conversion rate to biliverdin is approximately 4-fold slower than that by rat heme oxygenase (HO) isoform 1. Reaction of the hemin-Hmu O complex with hydrogen peroxide yields a verdoheme species, the recovery of which is much less compared with rat HO-1. Reaction of the hemin complex withmeta-chloroperbenzoic acid generates a ferryl oxo species. Thus, the catalytic intermediate species and the nature of the active form in the first oxygenation step of Hmu O appear to be similar to those of the mammalian HO. However, the considerably slow catalytic rate and low level of verdoheme recovery in the hydrogen peroxide reaction suggest that the active-site structure of Hmu O is different from that of its mammalian counterpart.

Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase. Properties of the heme and enzyme complex.

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform‐1, Syn HO‐1, has been characterized, while isoform‐2, Syn HO‐2, has not. In this study, a full‐length ho2 gene was cloned using synthetic DNA and Syn HO‐2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO‐1, the purified Syn HO‐2 bound hemin stoichiometrically to form a heme–enzyme complex and degraded heme to biliverdin IXα, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO‐2 was found to be comparable to that of Syn HO‐1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO‐2 converted heme to verdoheme. This shows that during the conversion of hemin to α‐meso‐hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin–Syn HO‐2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin‐Syn HO‐1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa‐coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO‐2 is different from that of Syn HO‐1.

Detection of Hepatitis G virus RNA in patients with Hepatitis B, Hepatitis C, and non-A-E Hepatitis by RT-PCR using multiple primer sets

Hepatitis G virus(HGV)/GB virus C(GBV‐C) is a newly identified virus associated with human hepatitis. The preliminary prevalence studies of HGV infection in Japan were entirely based on the detection of HGV RNA by RT‐PCR. However, the selection of the different primer sets in such assay may influence sensitivity of the test because of the extensive genetic heterogeneity of HGV, and influence the estimation of the prevalence of HGV. To address this potential problem, we designed two primer sets from well conserved domains in the 5′NC and NS5 regions of HGV genome, and tested them together with the NS3‐derived primer set in RT‐PCR for their ability to detect HGV RNA in serial dilution of synthetic viral RNA templates. Subsequently, we used these three primer sets to detect HGV RNA in the sera of 371 Japanese patients with hepatitis B, hepatitis C, and non‐A‐E hepatitis. The results indicated that the primer set derived from the 5′NC region appeared to be most effective in detecting HGV RNA. The results also showed that only two out of the 126 patients (1.6%) with non‐A‐E hepatitis were positive for HGV RNA although the RNA were detected more frequently in patients with hepatitis B (2/38; 5.3%) and hepatitis C (17/207; 8.2%), suggesting that HGV is not a common causative agent for non‐A‐E hepatitis in Japan. J. Med. Virol. 52:385–390, 1997.

Histidine 20, the Crucial Proximal Axial Heme Ligand of Bacterial Heme Oxygenase Hmu O from Corynebacterium diphtheriae

The hemin complex of Hmu O, a 24-kDa soluble heme degradation enzyme in Corynebacterium diphtheriae, is coordinated axially to a neutral imidazole of a proximal histidine residue in Hmu O. To identify which of the eight histidines in Hmu O is the proximal heme ligand, we have constructed and expressed the plasmids for eight His → Ala Hmu O mutants. Reconstituted with hemin, the active site structures and enzymatic activity of these mutants have been examined by EPR, resonance Raman, and optical absorption spectroscopy. EPR of the NO-bound ferrous heme-Hmu O mutant complexes reveals His20 as the proximal heme ligand in Hmu O, and this is confirmed by resonance Raman results from the ligand-free ferrous heme-H20A. All eight His → Ala mutants bind hemin stoichiometrically, proving that none of the histidines is essential for hemin-Hmu O formation. However, His20 is crucial to Hmu O catalysis. Its absence by point mutation has inhibited the conversion of hemin to biliverdin. The ferric heme-H20A complex is pentacoordinate. Resonance Raman of the CO-bound ferrous heme-H20A corroborates this and reveals an Fe-C-O bending mode, δ(Fe-C-O), the first reported for a pentacoordinate CO-bound hemeprotein. The appearance of δ(Fe-C-O) in C. diphtheriae Hmu O H20A but not mammalian HO-1 mutant H25A indicates that the heme environment between the two heme oxygenases is different.

Spectroscopic characterization of a higher plant heme oxygenase isoform-1 from Glycine max (soybean) −coordination structure of the heme complex and catabolism of heme

Heme oxygenase converts heme into biliverdin, CO, and free iron. In plants, as well as in cyanobacteria, heme oxygenase plays a particular role in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IXα as a direct synthetic resource. In this study, a higher plant heme oxygenase, GmHO‐1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion. The similarity in the amino acid sequence between GmHO‐1 and heme oxygenases from other biological species is low, and GmHO‐1 binds heme with 1 : 1 stoichiometry at His30; this position does not correspond to the proximal histidine of other heme oxygenases in their sequence alignments. The heme bound to GmHO‐1, in the ferric high‐spin state, exhibits an acid–base transition and is converted to biliverdin IXα in the presence of NADPH/ferredoxin reductase/ferredoxin, or ascorbate. During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme–heme oxygenase or CO–verdoheme–heme oxygenase complexes was observed and was extracted as a bis‐imidazole complex; it was identified as verdoheme. A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation. Thus, the mechanism of heme degradation by GmHO‐1 appears to be similar to that of known heme oxygenases, despite the low sequence homology. The heme conversion by GmHO‐1 is as fast as that by SynHO‐1 in the presence of NADPH/ferredoxin reductase/ferredoxin, thereby suggesting that the latter is the physiologic electron‐donating system.

Relevant expression of Drosophila heme oxygenase is necessary for the normal development of insect tissues

Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in micro-organisms, insects, plants, and mammals. The mammalian enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. The present study reports the use of RNA-interference (RNAi) to suppress HO in the multicellular eukaryote Drosophila. Eye imaginal disc-specific suppression of the Drosophila HO homolog (dHO) conferred serious abnormal eye morphology in adults. Deficiency of the dHO protein resulted in increased levels of iron and heme in larvae. The accumulation of iron was also observed in the compound eyes of dHO-knockdown adult flies. In parallel with the decrease of dHO, the expression of delta-aminolevulinic acid synthase, the first enzyme of the heme-biosynthetic pathway, in larvae was decreased markedly, suggesting that heme biosynthesis was totally suppressed by dHO-deficiency. The activation of caspase-3 occurred in eye imaginal discs of dHO-knockdown flies, indicating the occurrence of apoptosis in the discs. On the other hand, the overexpression of dHO resulted in a weak but significant rough eye phenotype in adults. Taken together, considering that dHO is not a stress-inducible protein, the expression of dHO can be tightly regulated at developmental stages and the relevant expression is necessary for the normal development of tissues in Drosophila.

Diversity of hepatitis G virus within a single infected individual.

The extent of population diversity among GB virus C (GBV-C)/hepatitis G virus (HGV) within a persistently infected individual (Iw) was investigated by sequence analysis of multiple clones generated from polymerase chain reaction (PCR)-amplified products of cDNA analogous to fragments of 5′ non-coding region (5′NC), envelope region 1/2 (E1/E2) and non-structural region 3 (NS3) of viral genome. Although nucleotide substitutions were more common in coding regions than in the 5′NC region, there was no region corresponding to the hypervariable region of hepatitis C virus in the E1/E2 region. Transition substitution exceeded transversion by 7 to 12-fold, and 79.4% of substitutions were synonymous. This bias against substitutions producing amino acid replacements and the use of Pfu DNA polymerase with an error rate 10 times lower than the observed frequency of substitution, suggests that most substitutions were not artefactual. This data suggests that individual genomes of HGV within an infected individual may differ from each other at 0.23–0.84% nucleotide position and at 0.42–0.61% amino acid position.

1H NMR Study of the Influence of Mutation on the Interaction of the C-Terminus with the Active Site in Heme Oxygenase from Neisseria meningitidis: Implications for Product Release

The HO from the pathogenic bacterium Neisseria meningitidis, NmHO, possesses C-terminal His207, Arg208, and His209 residues that are undetected in crystal structures. NMR found the C-terminus ordered and interacting with the active site and shown to undergo a spontaneous cleavage of the C-terminal Arg208-His209 bond that affects the product off rate. A preliminary model for the interaction based on the wild-type (WT) NmHO complexes has been presented [Liu, Y., Ma, L.-H., Satterlee, J. D., Zhang, X., Yoshida, T., and La Mar, G. N. (2006) Biochemistry 45, 3875-3886]. Two-dimensional (1)H NMR data of resting-state, azide-inhibited substrate complexes of the three C-terminal truncation mutants (Des-His209-, Des-Arg208His209-, and Des-His207Arg208His209-NmHO) confirm the previous proposed roles for His207 and Arg208 and reveal important additional salt bridges involving the His209 carboxylate and the side chains of both Lys126 and Arg208. Deletion of His209 leads to a qualitatively retained C-terminal geometry, but with increased separation between the C-terminus and active site. Moreover, replacing vinyls with methyls on the substrate leads to a decrease in the separation between the C-terminus and the active site. The expanded model for the C-terminus reveals a less stable His207-Arg208 cis peptide bond, providing a rationalization for its spontaneous cleavage. The rate of this spontaneous cleavage is shown to correlate with the proximity of the C-terminus to the active site, suggesting that the closer interaction leads to increased strain on the already weak His207-Arg208 peptide bond. The relevance of the C-terminus structure for in vitro studies, and the physiological function of product release, is discussed.

1H NMR study of the effect of variable ligand on heme oxygenase electronic and molecular structure

Heme oxygenase carries out stereospecific catabolism of protohemin to yield iron, CO and biliverdin. Instability of the physiological oxy complex has necessitated the use of model ligands, of which cyanide and azide are amenable to solution NMR characterization. Since cyanide and azide are contrasting models for bound oxygen, it is of interest to characterize differences in their molecular and/or electronic structures. We report on detailed 2D NMR comparison of the azide and cyanide substrate complexes of heme oxygenase from Neisseria meningitidis, which reveals significant and widespread differences in chemical shifts between the two complexes. To differentiate molecular from electronic structural changes between the two complexes, the anisotropy and orientation of the paramagnetic susceptibility tensor were determined for the azide complex for comparison with those for the cyanide complex. Comparison of the predicted and observed dipolar shifts reveals that shift differences are strongly dominated by differences in electronic structure and do not provide any evidence for detectable differences in molecular structure or hydrogen bonding except in the immediate vicinity of the distal ligand. The readily cleaved C-terminus interacts with the active site and saturation-transfer allows difficult heme assignments in the high-spin aquo complex.

Epidemiological study and genetic analysis of GB virus C infection in general population from an area endemic for hepatitis C.

The aim of this work was to study the prevalence, potential risk factors, clinical and laboratory features of GB virus C (GBV‐C) infection in general population from an area endemic for hepatitis C. A reverse transcriptase‐polymerase chain reaction (RT‐PCR) for detection of GBV‐C RNA was used to examine the prevalence of GBV‐C RNA in both hepatitis C virus (HCV) endemic (R town) and nonendemic areas (M town) in Yamagata prefecture, Japan. In R town, GBV‐C RNA was detected in 23 (2.9%) out of the 800 residents, whereas anti‐HCV and HCV‐RNA were found in 226 (28.3%) and 163 (20.4%), respectively. The prevalence of GBV‐C RNA in R town (2.9%) was higher than that in M town (1.0%), although the difference was not statistically significant. The individuals with anti‐HCV had significantly higher frequency of active GBV‐C infection than those without anti‐HCV in both towns. No evidence indicating that GBV‐C infection affected the severity of hepatitis C was obtained. The multivariate analysis revealed that the young anti‐HCV positive individuals with a history of blood transfusion had higher incidence of active GBV‐C infection. The phylogenetic analysis showed that the GBV‐C isolates from both R and M towns were divided into two separate branch groups designated HG and Asia GB groups. J. Med. Virol. 54:237–242, 1998.