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Examines the interaction of heme oxygenase-2 (HO-2) with nitric oxide (NO) donors. Absence of HO-2 heme regulatory motifs in HO-1 and HO-2 catalytic activity; Reactivity of HO isozymes with NO species produced by NO donors; Production of peroxynitrite by 3-morpholinosydnonimine.

Biliverdin reductase is unique among all enzymes described to date in having two pH optima, 6.75 and 8.7. at which NADH or NADPH, respectively, are required for activity. The enzyme converts biliverdin to bilirubin in mammals. The mature enzyme, which is 293 amino acids long, has 3 cysteine residues, and is sulfhydryl dependent. To understand the role of the cysteine residues in enzyme activity, we examined the effects of the neutral substitution with alanine of each of three residues, individually and in combination, by site-directed mutagenesis. These residues in the predicted amino acid sequence of rat biliverdin reductase correspond to amino acids 73. 280 and 291. The modification of the amino-proximal cysteine (Cys73), which is flanked by a tyrosine residue. completely inactivated the enzyme with NADH at pH 6.75 and NADPH at pH 8.7. The loss of reductase activity was not due to changes in three-dimensional characteristics of the protein as suggested by its mobility in a non-denaturing gel. Although modification of either of the two cysteines located near the C-terminus (Cys280 and Cys291) significantly reduced activity with both cofactors, these mutations did not inactivate the enzyme. Comparison of Km values for the Cys280→Ala and Cys291→Ala mutants with the wild type protein, at pH 8.7. suggests that Cys280 principally functions in substrate binding while Cys291 is predominantly involved in cofactor binding. This assignment probably also applies at pH 6.75. Comparison of kcat of the mutants with wild type shows that mutation of Cys280 decreases Vmax of the enzyme. Mutation of both C-terminal cysteines caused inactivation of the enzyme, comparable to that produced by mutation of Cys73. Analysis by circular dichroism at far-ultraviolet wavelengths suggests that the alterations in activity are not the result of changes in the secondary structure of these mutants. These results are consistent with Cys73 having a central role in substrate/cofactor binding while biliverdin reductase can function, albeit at a reduced rate, with only one of the near C-terminus cysteines. The results are further consistent with the suggestion that although the two C-terminal cysteines have preferential affinities, they can serve similar functions in the interaction with sub- strate/cofactor. [ABSTRACT FROM AUTHOR]

Two isozymes of heme oxygenase (HO), HO-1 or HSP32 and the constitutive form HO-2, have been characterized to date. We report the discovery of a third protein species and refer to it as HO-3. HO-3 is the product of a single transcript of ≈2.4 kb and can encode a protein of ≈33 kDa. The HO-3 transcript is found in the spleen, liver, thymus, prostate, heart, kidney, brain and testis and is the product of a singlecopy gene. The predicted amino acid structure of HO-3 differs from both HO-1 (HSP32) and HO-2 but is closely related to HO-2 (≈90%). Escherichia coli expressed and purified HO-3 protein does not cross react with polyclonal antibodies to either rat HO-1 or HO-2, is a poor heme catalyst, and displays hemoprotein spectral characteristics. The predicted protein has two heme regulatory motifs that may be involved in heme binding. These motifs and the hemoprotein nature of HO-3 suggest a potential regulatory role for the protein in cellular processes which are heme-dependent. [ABSTRACT FROM AUTHOR]

Biliverdin IXα reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin. BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH, NADPH) specificity A cDNA chine encoding human BVR was isolated from a &lamba; library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA. This approach was token because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed, The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296-amino-acid protein in which could be identified four peptides previously identified by micro-sequencing purified protein. The eDNA hybridized with a single message of ≈1.2 kb in human kidney poly(A)-rich RNA, and appeared, by Southern blot analysis, to be the product of a single-copy gene. Sequence analysis indicated that the human reductase shows ≈835 identity, at both the nucleotide and amino acid levels, with rat BVR. In some regions including the carboxyl terminus, protein sequence identity drops to 45%. Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat). The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence. However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rate reductase, which most likely utilizes truly NADPH under physiological conditions. Western blot analysis and isoelectric focusing demonstrate that, although migrating as a single band on SDS/PAGE. the expressed protein, like that purified from tissue, consists of several isoelectric charge variants. Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ration. That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli. Exogenous Zn also inhibits NADPH-dependent, but not NADH-dependent activity. Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn:Fe-.hematoporphyrin. however, inhibited both NADH- and NADPH-dependent activity. [ABSTRACT FROM AUTHOR]