Evidence that the catalytic mechanism of heme a synthase involves the formation of a carbocation stabilized by a conserved glutamate.

In eukaryotes and many aerobic prokaryotes, the final step of aerobic respiration is catalyzed by an aa3-type cytochrome c oxidase, which requires a modified heme cofactor, heme a. The conversion of heme b, the prototypical cellular heme, to heme o and ultimately to heme a requires two modifications, the latter of which is conversion of a methyl group to an aldehyde, catalyzed by heme a synthase (HAS). The N- and C-terminal halves of HAS share homology, and each half contains a heme-binding site. Previous reports indicate that the C-terminal site is occupied by a heme b cofactor. The N-terminal site may function as the substrate (heme o) binding site, although this has not been confirmed experimentally. Here, we assess the role of conserved residues from the N- and C-terminal heme-binding sites in HAS from prokaryotic (Shewanella oneidensis) and eukaryotic (Saccharomyces cerevisiae) species - SoHAS/CtaA and ScHAS/Cox15, respectively. A glutamate within the N-terminal site is found to be critical for activity in both types of HAS, consistent with the hypothesis that a carbocation forms transiently during catalysis. In contrast, the residue occupying the analogous C-terminal position is dispensable for enzyme activity. In SoHAS, the C-terminal heme ligands are critical for stability, while in ScHAS, substitutions in either heme-binding site have little effect on global structure. In both species, in vivo accumulation of heme o requires the presence of an inactive HAS variant, highlighting a potential regulatory role for HAS in heme o biosynthesis.

Loss of Num1-mediated cortical dynein anchoring negatively impacts respiratory growth.

Num1 is a multifunctional protein that both tethers mitochondria to the plasma membrane and anchors dynein to the cell cortex during nuclear inheritance. Previous work has examined the impact loss of Num1-based mitochondrial tethering has on dynein function in Saccharomyces cerevisiae; here, we elucidate its impact on mitochondrial function. We find that like mitochondria, Num1 is regulated by changes in metabolic state, with the protein levels and cortical distribution of Num1 differing between fermentative and respiratory growth conditions. In cells lacking Num1, we observe a reproducible respiratory growth defect, suggesting a role for Num1 in not only maintaining mitochondrial morphology, but also function. A structure-function approach revealed that, unexpectedly, Num1-mediated cortical dynein anchoring is important for normal growth under respiratory conditions. The severe respiratory growth defect in Δnum1 cells is not specifically due to the canonical functions of dynein in nuclear migration but is dependent on the presence of dynein, as deletion of DYN1 in Δnum1 cells partially rescues respiratory growth. We hypothesize that misregulated dynein present in cells that lack Num1 negatively impacts mitochondrial function resulting in defects in respiratory growth.

Mitochondrial contact site and cristae organizing system (MICOS) machinery supports heme biosynthesis by enabling optimal performance of ferrochelatase.

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied and its biosynthetic enzymes structurally characterized to varying extents. Nevertheless, understanding of the regulation of heme synthesis and factors that influence this process in metazoans remains incomplete. Therefore, we investigated the molecular organization as well as the physical and genetic interactions of the terminal pathway enzyme, ferrochelatase (Hem15), in the yeast Saccharomyces cerevisiae. Biochemical and genetic analyses revealed dynamic association of Hem15 with Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS). Loss of MICOS negatively impacts Hem15 activity, affects the size of the Hem15 high-mass complex, and results in accumulation of reactive and potentially toxic tetrapyrrole precursors that may cause oxidative damage. Restoring intermembrane connectivity in MICOS-deficient cells mitigates these cytotoxic effects. These data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme homeostasis.

A central core disease mutation in the Ca2+-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation.

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca2+-mediated activation of skeletal muscle Ca2+ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca2+ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca2+ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca2+ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca2+-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca2+-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca2+ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca2+ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca2+ of the two mutants, whereas the interaction of Ca2+ with directly interacting RyR1 amino acid residues Glu3893, Glu3967, and Thr5001 was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca2+-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.