Abstract
The integral membrane protein heme A synthase (HAS) catalyzes the biosynthesis of heme A, which is a prerequisite for cellular respiration in a wide range of aerobic organisms. Previous studies have revealed that HAS can form homo-oligomeric complexes, and this oligomerization appears to be evolutionarily conserved among prokaryotes and eukaryotes and is shown to be essential for the biological function of eukaryotic HAS. Despite its importance, little is known about the detailed structural properties of HAS oligomers. Here, we aimed to address this critical issue by analyzing the oligomeric state of HAS from Aquifex aeolicus (AaHAS) using a combination of techniques, including size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), cross-linking, laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), and single-particle electron cryomicroscopy (cryo-EM). Our results show that HAS forms a thermostable trimeric complex. A cryo-EM density map provides information on the oligomerization interface of the AaHAS trimer. These results provide structural insights into HAS multimerization and expand our knowledge of this important enzyme.IMPORTANCE Heme A is a vital redox cofactor unique for the terminal cytochrome c oxidase in mitochondria and many microorganisms. It plays a key role in oxygen reduction by serving as an electron carrier and as the oxygen-binding site. Heme A is synthesized from heme O by an integral membrane protein, heme A synthase (HAS). Defects in HAS impair cellular respiration and have been linked to various human diseases, e.g., fatal infantile hypertrophic cardiomyopathy and Leigh syndrome. HAS exists as a stable oligomeric complex, and studies have shown that oligomerization of eukaryotic HAS is necessary for its proper function. However, the molecular architecture of the HAS oligomeric complex has remained uncharacterized. The present study shows that HAS forms trimers and reveals how the oligomeric arrangement contributes to the complex stability and flexibility, enabling HAS to perform its catalytic function effectively. This work provides the basic understanding for future studies on heme A biosynthesis.
Highlights
The integral membrane protein heme A synthase (HAS) catalyzes the biosynthesis of heme A, which is a prerequisite for cellular respiration in a wide range of aerobic organisms
To determine the oligomeric state of AaHAS, blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis was conducted, and a single dominant band migrating at approximately 200 kDa was detected (Fig. 1B). This result is similar to that found in previous studies [13, 18], where COX15 from S. cerevisiae exists as an oligomeric complex as observed on BN-PAGE analysis
We investigated the oligomeric state of AaHAS using complementary methods
Summary
The integral membrane protein heme A synthase (HAS) catalyzes the biosynthesis of heme A, which is a prerequisite for cellular respiration in a wide range of aerobic organisms. The purified AaHAS eluted as a monodisperse peak which was monitored at both 280 and 415 nm, indicating the incorporation of heme cofactors (Fig. 1A). To determine whether heme O is incorporated, AaHAS was analyzed by high-performance liquid chromatography with UV detection coupled to mass spectrometry (Fig. S2).
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