Expression and Characterization of Full-Length Human Heme Oxygenase-1:  The Presence of Intact Membrane-Binding Region Leads to Increased Binding Affinity for NADPH Cytochrome P450 Reductase

Abstract

Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200-225 nmol bilirubin hr −1 nmol −1 HO-1 as compared to ~140-150 nmol bilirubin hr −1 nmol −1 for the WT form, which contains the 30-kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes.Heme oxygenase catalyzes the breakdown of heme to biliverdin, iron, and carbon monoxide (CO) (1). The physiological significance of these metabolites, most notably CO and biliverdin, is a reason for the escalating interest in HO research within the last 15 years (2). HO regulates iron homeostasis in mammals by recycling the iron released from the porphyrin ring. Biliverdin reductase, a soluble enzyme found in the cytosol, converts biliverdin to bilirubin. Bilirubin is a potent antioxidant that is conjugated to glucuronic acid in order to be excreted (3). Obstruction of bilirubin elimination has been implicated in common conditions such as neonatal jaundice, which can lead to neurological damage (4). CO, a third product of heme oxygenase, has been *Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology and the Stanley S. Scott Cancer Center, LSU Health Sciences Center, 533 Bolivar Street, New Orleans, La 70112, Voice 504-568-6557, FAX 504-568-6888, wbacke@lsuhsc.edu. shown to have signaling properties that parallel those of nitric oxide (5-7). CO is believed to elicit its effects via the guanylate cyclase pathway, common to NO. NIH Public AccessTwo disti...

Measurement of Membrane-Bound Human Heme Oxygenase-1 Activity Using a Chemically Defined Assay System

“…The membrane-binding region of full-length HO-1 has a significant influence on the kinetic characteristics of the enzyme (Huber and Backes, 2007). These differences call into question whether conditions that are adequate for measuring the activity of soluble HO-1 can be used to examine the full-length form of the enzyme.…”

“…HO-1 activity was examined using the compound bilirubin assay as previously reported (Maines, 1996;Huber and Backes, 2007) with minor modifications. Bilirubin formation was monitored at 464 to 530 nm, and the enzyme activity was calculated using the extinction coefficient 40 mM Ϫ1 cm…”

Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds

“…However, the recent characterization of a recombinant full-length HO-1 had indicated that the fulllength form exhibits a 2-to 3-fold greater activity relative to that of the truncated, soluble form, which was increased even further in the presence of lipid. Moreover, the C-terminal hydrophobic tail has an essential role with respect to membrane incorporation as well as in formation of a high-affinity complex with cytochrome P450 reductase, deemed essential for maximal catalytic activity [93][94][95]. The C-terminal domain is also the major point of sequence divergence between the two isozymes, especially in the presence of the HRMs in HO-2 [58].…”

Elucidating the Role of Biliverdin Reductase in the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress

“…One reason for this is the fact that the full-length HO-1 is very unstable and susceptible to truncation that generates an inactive, soluble form (28 kDa). The C-terminal part of the protein that is cleaved causes the HO-1 to interact with membrane lipids, and its removal alters the manner by which the enzyme interacts with potential membrane binding partners (Huber, III et al, 2009;Huber, III and Backes, 2007). Most in vitro studies of HO-1 have expressed and purified a modified, but active, 30 kDa form of the enzyme that lacks the C-terminal membrane-binding sequence and is soluble as a result.…”

“…Most in vitro studies of HO-1 have expressed and purified a modified, but active, 30 kDa form of the enzyme that lacks the C-terminal membrane-binding sequence and is soluble as a result. Our lab has recently modified the amino acid sequence of full-length HO-1 to remove a thrombin cleavage site in the C-terminal tale of HO-1 (Huber, III and Backes, 2007). This mutant is full-length and binds to lipid vesicles.…”