Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER) which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans.

Background: NO-induced conformational changes of guanylate cyclase were analyzed. Results: FRET experiments show a movement of two tryptophans toward a fluorescent substrate. Conclusion: Activation is transmitted to the catalytic domain by direct interaction of the heme binding domain and through the coiled-coil helix. Significance: The results help to advance understanding of the molecular events associated with activation of NOsGC.

BackgroundNitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β1-subunit. A splice variant (C-α1) of the α1-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61–128 of the α1-subunit are mandatory for quantitative heterodimerization implying that the C-α1-splice variant should lose its capacity to dimerize quantitatively.Methodology/Principal FindingsIn the current study we demonstrate preserved quantitative dimerization of the C-α1-splice by co-purification with the β1-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β1-subunit and the α1-subunit or the C-α1 variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α1/β1 and C-α1/β1 than the negative control. In addition we show that lack of the amino-terminus in the α1 splice variant directs it to a more oxidized subcellular compartment.Conclusions/SignificanceWe conclude that the amino-terminus of the α1-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.

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