scholarly journals Heme Gazing: Illuminating Eukaryotic Heme Trafficking, Dynamics, and Signaling with Fluorescent Heme Sensors

Biochemistry ◽  
2017 ◽  
Vol 56 (13) ◽  
pp. 1815-1823 ◽  
Author(s):  
David A. Hanna ◽  
Osiris Martinez-Guzman ◽  
Amit R. Reddi
Keyword(s):  
Heme Trafficking

scholarly journals Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

2021 ◽  
Author(s):  
David A Hanna ◽  
Courtney M Moore ◽  
Liu Liu ◽  
Xiaojing Yuan ◽  
Angela S Fleischhacker ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Physiological Function ◽  
Hek293 Cells ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Heme Oxygenases ◽  
Heme Trafficking ◽  
Ferrous Heme

Heme oxygenases (HO) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms: inducible HO-1, which is up-regulated in response to various stressors, including excess heme, and constitutive HO-2. While much is known about the regulation and physiological function of HO-1, comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is largely dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or over-expressed HO-2, and various HO-2 mutant alleles, we found that endogenous heme is too limiting to support HO-2 catalyzed heme degradation. Rather, we discovered that a novel role for HO-2 is to bind and buffer labile heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor in control of heme bioavailability. When heme is in excess, HO-1 is induced and both HO-2 and HO-1 can provide protection from heme toxicity by enzymatically degrading it. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with the labile heme pool being oxidized, thereby providing new insights into heme trafficking and signaling.

scholarly journals HEME Trafficking by the Cytochrome C Biogenesis Pathways

2018 ◽  
Vol 114 (3) ◽  
pp. 425a
Author(s):  
Molly C. Sutherland ◽  
Joshua M. Jarodsky ◽  
Robert G. Kranz
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Biogenesis ◽  
Heme Trafficking

scholarly journals The CcmC:Heme:CcmE Complex in Heme Trafficking and Cytochrome c Biosynthesis

2010 ◽  
Vol 401 (3) ◽  
pp. 350-362 ◽  
Author(s):  
Cynthia Richard-Fogal ◽  
Robert G. Kranz
Keyword(s):  
Cytochrome C ◽  
Heme Trafficking

scholarly journals Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

2016 ◽  
Vol 113 (27) ◽  
pp. 7539-7544 ◽  
Author(s):  
David A. Hanna ◽  
Raven M. Harvey ◽  
Osiris Martinez-Guzman ◽  
Xiaojing Yuan ◽  
Bindu Chandrasekharan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Glycolytic Enzyme ◽  
Unicellular Eukaryote ◽  
Human Cell Lines ◽  
State Distribution ◽  
Signaling Molecule ◽  
Nuclear Transcription Factor ◽  
Heme Acquisition ◽  
Oxidation State Distribution ◽  
Heme Trafficking

Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryoteSaccharomyces cerevisiae. We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20–40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, includingEscherichia coli, yeast, and human cell lines.

scholarly journals Structure-Function Analysis of the Bifunctional CcsBA Heme Exporter and CytochromecSynthetase

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Molly C. Sutherland ◽  
Nathan L. Tran ◽  
Dustin E. Tillman ◽  
Joshua M. Jarodsky ◽  
Jason Yuan ◽  
...  
Keyword(s):  
Structure Function ◽  
Binding Site ◽  
Binding Sites ◽  
Structural Model ◽  
Function Analysis ◽  
Integral Membrane Protein ◽  
Covalent Attachment ◽  
Heme Binding ◽  
Heme Transport ◽  
Heme Trafficking

ABSTRACTAlthough intracellular heme trafficking must occur for heme protein assembly, only a few heme transporters have been unequivocally discovered and nothing is known about their structure or mechanisms. Cytochromecbiogenesis in prokaryotes requires the transport of heme from inside to outside for stereospecific attachment to cytochromecvia two thioether bonds (at CXXCH). The CcsBA integral membrane protein was shown to transport and attach heme (and thus is a cytochromecsynthetase), but the structure and mechanisms underlying these two activities are poorly understood. We employed a new cysteine/heme crosslinking tool that traps endogenous heme in heme binding sites. We combined these data with a comprehensive imidazole correction approach (for heme ligand interrogation) to map heme binding sites. Results illuminate the process of heme transfer through the membrane to an external binding site (called the WWD domain). Using meta-genomic data (GREMLIN) and Rosetta modeling programs, a structural model of the transmembrane (TM) regions in CcsBA were determined. The heme mapping data were then incorporated to model the TM heme binding site (with TM-His1 and TM-His2 as ligands) and the external heme binding WWD domain (with P-His1 and P-His2 as ligands). Other periplasmic structure/function studies facilitated modeling of the full CcsBA protein as a framework for understanding the mechanisms. Mechanisms are proposed for heme transport from TM-His to WWD/P-His and subsequent stereospecific attachment of heme. A ligand exchange of the P-His1 for histidine of CXXCH at the synthetase active site is suggested.IMPORTANCEThe movement or trafficking of heme is critical for cellular functions (e.g., oxygen transport and energy production); however, intracellular heme is tightly regulated due to its inherent cytotoxicity. These factors, combined with the transient nature of transport, have resulted in a lack of direct knowledge on the mechanisms of heme binding and trafficking. Here, we used the cytochromecbiogenesis system II pathway as a model to study heme trafficking. System II is composed of two integral membrane proteins (CcsBA) which function to transport heme across the membrane and stereospecifically position it for covalent attachment to apocytochromec. We mapped two heme binding domains in CcsBA and suggest a path for heme trafficking. These data, in combination with metagenomic coevolution data, are used to determine a structural model of CcsBA, leading to increased understanding of the mechanisms for heme transport and the cytochromecsynthetase function of CcsBA.

scholarly journals Heme Trafficking by the Cytochrome C Biogenesis Pathways

2017 ◽  
Vol 112 (3) ◽  
pp. 65a-66a
Author(s):  
Molly C. Sutherland ◽  
Joel A. Rankin ◽  
Robert G. Kranz
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Biogenesis ◽  
Heme Trafficking
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