Abstract
Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.
Highlights
In 1968 as an enzyme catalyzing the oxidative cleavage of heme in mammalian microsomes [1, 2]
Based on the amino acid sequence alignment, Glycine max (soybean) Heme oxygenase (HO)-1 (GmHO-1) lacks some essential amino acid residues, including Asp140, which is a key residue in a hydrogenbonding network for proton transfer, and His25, which coordinates with heme, Gohya et al [31] previously observed ligation of heme from His in the spectroscopic measurement of GmHO-1
We compared the electrostatic potentials of HmuO and human HO isozyme1 (hHO-1) mapped on the molecular surface, which showed that the positively charged character of the surface near the bound heme is common to all three HOs, but only GmHO-1 has a dented cave-like surface to accommodate the small globular Fd molecule (Fig. S1)
Summary
We tried to solve the crystal structure of GmHO-1 by the molecular replacement method using published HO structures as a search model, but this approach failed, implying a significant structural difference between plant-type and other HOs. The heme molecule bound to GmHO-1 is sandwiched between proximal (α1) and distal (α5) helices with axial ligation from His residue (Fig. 1B), consistent with previous data from electron paramagnetic resonance measurements [31]. Superposing the structures of GmHO-1 with hHO-1 and HmuO clarifies similarities and differences between plant-type HO and other HOs (Fig. 1D). Plant-type specific interactions with heme are mediated by five residues (Glu, Ala149, Phe214, Ser217, and Leu221) (Fig. 3B), all of which make hydrophobic contacts with the heme molecule These five residues are located near the newly identified structural features of the irregularly structured region and curved α7 helix and probably avert conflict between heme binding and these novel structural features. HmuO, a gene oxygenase of Corynebacterium diphtheriae; HO-1, heme oxygenase 1
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