Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase

Abstract

The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPRsupported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling. KeywordsHeme oxygenase; NADPH-cytochrome P450 reductase; biliverdin reductase; MALDI-TOF mass spectrometry; chemical modification; protein-protein interaction Heme oxygenase (HO, EC 1.14.99.3) is a membrane-bound microsomal enzyme that catalyzes the degradation of heme to biliverdin, carbon monoxide (CO), and free iron [1,2]. Biliverdin is subsequently converted to bilirubin by a soluble cytosolic enzyme, biliverdin IXα reductase (BVR, EC 1.2.1.24) [3]. The electrons required for HO catalysis in mammals are provided by NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4), a 78-kDa membrane-bound * Correspondence should be addressed to: Prof. Masato Noguchi, Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan, Tel.: +81-942-31-7544; fax: +81-942-31-4377. E-mail address: mnoguchi@med.kurume-u.ac.jp (M. Noguchi).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Recently, using site-specific mutation and docking modeling, we found that Arg-185 and Lys-149 of rat HO-1 are important in both the HO activity and its association with CPR [16]. In our model, the guanidino group of Arg-185 interacts electrostatically with 2′-phosphate of NADPH bound to CPR. On the other hand, Lys-149 is close to the acidic amino acid clusters near the FMN binding site of CPR; mutation of the acidic clusters causes reduction of the electron transfer rates from CPR to cytochrome P450s [17]. Thus, Arg-185 and Lys-149 appear to interact with CPR in such a way as to orient the redox partners for optimal electron transfer from CPR to the heme of HO-1. The model also suggested that the electrons from CPR are ...

New Insights into Intracellular Locations and Functions of Heme Oxygenase-1

“…Lysine acetylation has also been shown to be important for cell cycle, nuclear transport, and chromatin remodeling. When assessing the residues that are important for the interaction between rat HMOX1 and CPR, nine (i.e., K18, K22, K39, K48, K69, K149, K153, K179, and K196) out of a total of 15 lysine residues were identified by mass spectrometry (MAL-DI-TOF) as acetylated (61). Interestingly, K149 and K153 were protected from acetylation in the presence of CPR.…”

Protein/Protein Interactions in the Mammalian Heme Degradation Pathway

“…These reports provide K d values for the HO-1⅐CPR complex that range from 0.4 Ϯ 0.1 to 2.4 Ϯ 0.6 M. CPR protects lysine residues 149 and 153 in HO-1 from chemical modification, suggesting that these basic residues are at the interface of the HO-1⅐CPR complex (25). Additionally, SPR studies indicate that the K149A substitution results in an ϳ10-fold increase in K d for the HO-1⅐CPR complex (24).…”

“…The complex between the soluble forms of CPR and HO-1 (lacking their membrane-spanning regions) has been characterized by fluorescence quenching (27), surface plasmon resonance (SPR) (24), and acetylation protection assays (25). These reports provide K d values for the HO-1⅐CPR complex that range from 0.4 Ϯ 0.1 to 2.4 Ϯ 0.6 M. CPR protects lysine residues 149 and 153 in HO-1 from chemical modification, suggesting that these basic residues are at the interface of the HO-1⅐CPR complex (25).…”

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

“…Covalent labeling (CL-MS) [9], using reactive probes to identify the chemically-accessible surface areas (CASA) of a protein, can generate a form of topographical map very useful for protein characterization. In the context of intermolecular interactions, this can support the localization of binding surfaces [10][11][12]. Label data can even constrain 3D protein structure prediction [13].…”