Spectroscopic and mutagenesis studies of human PGRMC1

Abstract

Progesterone receptor membrane component 1 (PGRMC1) is a 25 kDa protein with an N-terminal transmembrane domain and a putative C-terminal cytochrome b5 domain. Heme-binding activity of PGRMC1 has been shown in various homologues of PGRMC1. Although the general definition of PGRMC1 is as a progesterone receptor, progesterone-binding activity has not been directly demonstrated in any of the purified PGRMC1 proteins fully loaded with heme. Here, we show that the human homologue of PGRMC1 (hPGRMC1) binds heme in a five-coordinate (5C) high-spin (HS) configuration, with an axial tyrosinate ligand, likely Y95. The negatively charged tyrosinate ligand leads to a relatively low redox potential of approximately -331 mV. The Y95C or Y95F mutation dramatically reduces the ability of the protein to bind heme, supporting the assignment of the axial heme ligand to Y95. On the other hand, the Y95H mutation retains ∼90% of the heme-binding activity. The heme in Y95H is also 5CHS, but it has a hydroxide axial ligand, conceivably stabilized by the engineered-in H95 via an H-bond; CO binding to the distal ligand-binding site leads to an exchange of the axial ligand to a histidine, possibly H95. We show that progesterone binds to hPGRMC1 and introduces spectral changes that manifest conformational changes to the heme. Our data offer the first direct evidence supporting progesterone-binding activity of PGRMC1.

Figure 1

Linear Nernst plots based on one-electron reduction of WT (A) and Y95H mutant (B) of hPGRMC1 and two-electron reduction of a dye, safranin T. P_ox/P_red and D_ox/D_red stand for the ratio of oxidized protein vs reduced protein and the ratio of oxidized dye vs reduced dye, respectively. The solid lines are the best fit lines with the slopes fixed to be 1. The redox potentials of the WT and Y95H mutant of hPGRMC are estimated based on the intercepts of the lines to be ~−331 and −284 mV, respectively, with the assumption that E° (safranin T) = −289 mV (vs SHE).

Figure 2

UV–vis (A) and high-frequency RR (B) spectra of WT hPGRMC1. The UV and RR spectra were obtained with ~5 and 80 µM protein, respectively, in 100 mM pH 7.4 potassium phosphate buffer.

Figure 3

Low-frequency RR spectra (A) and CO isotope-sensitive ν_Fe–CO/ν_C–O modes of WT hPGRMC1 (B). ν_Fe–CO vs ν_C–O inverse correlation plot (C). In (C), the data points associated with P450s (L = Cys), myoglobin variants (l-His), and 5C heme are taken from the literature^,,,, and are presented by black squares, circles, and triangles, respectively; those associated with PGRMC1, HasA, and H25Y mutant of hHO-1 are as indicated. The data were obtained with ~80 µM protein in 100 mM pH 7.4 potassium phosphate buffer.

Figure 4

UV–vis (A), high-frequency RR (B), low-frequency RR (D) spectra, and ν_Fe–CO and ν_C–O modes (C) of the Y95H mutant of hPGRMC1. The UV and RR spectra were obtained with ~5 and 80 µM protein, respectively, in 100 mM pH 7.4 potassium phosphate buffer.

Figure 5

UV–vis (A, B) and RR (C) spectra of the WT and Y95H mutant of ferric hPGRMC1 with (red) and without (black) progesterone. The concentrations of protein/progesterone are ~5/50 and 80/500 µM (in 100 mM pH 7.4 potassium phosphate buffer) for the UV–vis and RR measurements, respectively.

Figure 6

UV–vis (A, B) and RR (C) spectra of the WT and Y95H mutant of deoxy ferrous hPGRMC1 with (red) and without (black) progesterone. The concentrations of protein/progesterone are ~5/50 and 80/500 µM (in 100 mM pH 7.4 potassium phosphate buffer) for the UV–vis and RR measurements, respectively.

Scheme 1

Postulated Coordination States of the WT and Y95H Mutant of hPGRMC1