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/CPR-supported 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.

Fig. 1. Absorption spectral changes of the heme-rat HO-1 complex during the NADPH/CPR-supported single-turnover reaction

The spectra were recorded before (dotted line; ········) and after the addition of NADPH; 5 (dashed and dotted line; -·-·-), 10 (dashed line; ------) and 30 (solid line; —) min. (A) Unmodified HO-1, (B) HO-1 acetylated in the absence of CPR, (C) HO-1 acetylated in the presence of CPR. All reaction mixtures contained 5 μM heme-HO-1 complex, 40 nM CPR and 25 μM NADPH in 0.1M potassium phosphate buffer (pH 7.4).

Fig. 2. Mass spectrometric analysis of the heme-rat HO-1 complex

(A) MALDI-TOF mass spectra of the tryptic peptides from rat HO-1: (a) unmodified, (b) acetylated in the absence of the reductases, (c) acetylated in the presence of CPR, (d) acetylated in the presence of BVR. The 16 solid arrows in (a) indicate the positions of the peptide fragment derived from the unmodified HO-1. The dashed arrows, 9 in (b) and (d) and 7 in (c) indicate the peaks of the acetylated peptides. The single solid-line arrows at m/z 1249 in (b), (c) and (d) indicate the Tyr-137-Lys-148 peptide without acetylation. In (c), the peak at m/z 1377 (asterisk) indicates the Tyr-137-Lys-149 peptide without acetylation and the 2 hollow arrows indicate the peaks, m/z 1419 and 1902, which were not detected in HO-1 acetylated in the presence of CPR. (B) Sequence and secondary structure of rat HO-1. Identified amino acid sequences in the mass spectra from heme-unmodified rat HO-1 complex are indicated by bold characters. The α-helices are shown as cylinders.

Fig. 3. Putative docking model of rat HO-1 (PDB 1DVE) with rat BVR (1GCU) (A) and with CPR (1AMO) (B; ref 16) calculated by the program Hex

HO-1 is represented by a ribbon (purple). Heme is shown as ball-and-sticks (olive). The side chains of Lys-149 and Lys-153 in F helix of HO-1 are represented in stick form (red). BVR and CPR are shown as orange and green ribbons, respectively. The cofactors of CPR are in stick form (blue).