Ferritin protein is involved in biological tissues in the storage and management of iron - an essential micro-nutrient in the majority of living systems. While there are extensive studies on ...iron-loaded ferritin, its functionality in iron delivery is not completely clear. Here, for the first time, differential pulse voltammetry (DPV) has been successfully adapted to address the challenge of resolving a cascade of fast and co-occurring redox steps in enzymatic systems such as ferritin. Using DPV, comparative analysis of ferritins from two evolutionary-distant organisms has allowed us to propose a stepwise resolution for the complex mix of concurrent redox steps that is inherent to ferritins and to fine-tune the structure-function relationship of each redox step. Indeed, the cyclic conversion between Fe
and Fe
as well as the different oxidative steps of the various ferroxidase centers already known in ferritins were successfully discriminated, bringing new evidence that both the 3-fold and 4-fold channels can be functional in ferritin.
Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of ...intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.
Green Fluorescent Proteins (GFPs) are a family of proteins with a disjunct systematic distribution; their biological functions remain speculative for the most part. Here we report studies of 3 ...closely related species of green sea anemones (Anthopleura) that express GFPs throughout their ectoderm. Individuals of these species maintain facultative symbiosis with zooxanthellae in their endoderm and inhabit the rocky intertidal or shallow subtidal. Thus, they depend on exposure to light to maintain photosynthesis of their symbionts, and simultaneously need to manage stresses associated with this exposure. We present experimental evidence that these sea anemones regulate the amount of GFP in their bodies in response to the surrounding light environment: they increase or reduce GFP when exposed to brighter or dimmer light, respectively, yet they maintain some GFP while in darkness, for surprisingly long periods.
Ferritin, a multimeric cage-like enzyme, is integral to iron metabolism across all phyla through the sequestration and storage of iron through efficient ferroxidase activity. While ferritin sequences ...from ∼900 species have been identified, crystal structures from only 50 species have been reported, the majority from bacterial origin. We recently isolated a secreted ferritin from the marine invertebrate
sp. (parchment tube worm), which resides in muddy coastal seafloors. Here, we present the first ferritin from a marine invertebrate to be crystallized and its biochemical characterization. The initial ferroxidase reaction rate of recombinant
ferritin (ChF) is 8-fold faster than that of recombinant human heavy-chain ferritin (HuHF). To our knowledge, this protein exhibits the fastest catalytic performance ever described for a ferritin variant. In addition to the high-velocity ferroxidase activity, ChF is unique in that it is secreted by
in a bioluminescent mucus. Previous work has linked the availability of Fe
to this long-lived bioluminescence, suggesting a potential function for the secreted ferritin. Comparative biochemical analyses indicated that both ChF and HuHF showed similar behavior toward changes in pH, temperature, and salt concentration. Comparison of their crystal structures shows no significant differences in the catalytic sites. Notable differences were found in the residues that line both 3-fold and 4-fold pores, potentially leading to increased flexibility, reduced steric hindrance, or a more efficient pathway for Fe
transportation to the ferroxidase site. These suggested residues could contribute to the understanding of iron translocation through the ferritin shell to the ferroxidase site.
The natural phenomenon of bioluminescence is light production fueled by biochemical processes. In general, bioluminescence is observed as very short (millisecond) flashes, or a lasting glow in ...bacteria and mushrooms. However, the marine tube worm Chaetopterus sp., secretes a mucus that bioluminesces for a long time, up to 72 hours in seawater. The biochemical mechanisms associated with this process are progressively being understood, with the incentive of a variety of promising applications.
In our efforts to elucidate the mechanism behind this long‐lasting bioluminescence we consistently measure a significant light increase upon addition of Fe2+ to the secreted mucus. Considering an iron‐redox as possible source of energy, we found ferritin in abundance in the mucus, which could ultimately be the energy provider or “battery” of the luminous reaction.
We have isolated, sequenced, purified and crystallized this ferritin and found it to be up to 8 times faster than human heavy chain ferritin for its ferroxidase activity. A combination of electrochemistry measurements and structural analysis has led us to believe that this ferritin is the first natural ferritin described to use a different pathway to optimize its enzymatic activity.
Support or Funding Information
This research is funded by the Air Force Office of Scientific Research (grant number FA9550‐17‐0189 to D.D.D.)
Bioluminescence process in marine tube worm Chaetopterus is believed to be driven by Fe2+ released from ferritin in the mucus.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
An increasing number of luminous organisms are being found in the ocean, presenting opportunities to discover new biochemical reactions associated with light production, and thus new ...bioreporter systems for biotechnology and biomedicine. The marine parchment tube worm
Chaetopterus sp
. is of particular interest because it secretes a luminous mucus that glows for a long time (hours, possibly days), although in seawater and isolated from any continuous metabolic support. This contrasts with the short flashes, or the bacterial, metabolically driven glow that are more typical for naturally observed bioluminescence. We have recently found that the light production in this mucus is activated by Fe
2+
and that the long‐lasting glow may therefore be supported by the proven presence of the iron storage protein ferritin.
We have also observed a temporary increase in the bioluminescence after exposure of the mucus to blue light specifically, which is capable of triggering Fe
2+
release from ferritin through a photo‐reduction process. Since the mucus bioluminesces in the blue region of visible light, we are investigating whether it could be powering its own light production.
Support or Funding Information
This research is supported by the Air Force Office of Scientific Research (grant # FA955017 0189 to D. D. Deheyn).
The first hyperpolarizability (β) of six fluorescent proteins (FPs), namely, enhanced green fluorescent protein, enhanced yellow fluorescent protein, SHardonnay, ZsYellow, DsRed, and mCherry, has ...been calculated to unravel the structure–property relationships on their second-order nonlinear optical properties, owing to their potential for multidimensional biomedical imaging. The ONIOM scheme has been employed and several of its refinements have been addressed to incorporate efficiently the effects of the microenvironment on the nonlinear optical responses of the FP chromophore that is embedded in a protective β-barrel protein cage. In the ONIOM scheme, the system is decomposed into several layers (here two) treated at different levels of approximation (method1/method2), from the most elaborated method (method1) for its core (called the high layer) to the most approximate one (method2) for the outer surrounding (called the low layer). We observe that a small high layer can already account for the variations of β as a function of the nature of the FP, provided the low layer is treated at an ab initio level to describe properly the effects of key H-bonds. Then, for semiquantitative reproduction of the experimental values obtained from hyper-Rayleigh scattering experiments, it is necessary to incorporate electron correlation as described at the second-order Møller–Plesset perturbation theory (MP2) level as well as implicit solvent effects accounted for using the polarizable continuum model (PCM). This led us to define the MP2/6-31+G(d):HF/6-31+G(d)/IEFPCM scheme as an efficient ONIOM approach and the MP2/6-31+G(d):HF/6-31G(d)/IEFPCM as a better compromise between accuracy and computational needs. Using these methods, we demonstrate that many parameters play a role on the β response of FPs, including the length of the π-conjugated segment, the variation of the bond length alternation, and the presence of π-stacking interactions. Then, noticing the small diversity of the FP chromophores, these results highlight the key role of the β-barrel and surrounding residues on β, not only because they can locally break the noncentrosymmetry vital to a β response but also because it can impose geometrical constraints on the chromophore.
Abstract only
The marine polychaete worm
Chaetopterus
sp. secretes a sticky mucus that exhibits a soft blue long‐lasting bioluminescence. Iron (both ferrous and ferric) and riboflavin have been found ...abundant in the mucus and identified as potential cofactors involved in the control of the light production. The Deheyn lab has recently identified a novel ferritin protein (ChF) from fractions of the worm mucus still able to produce bioluminescence after purification by chromatography. Ferritin proteins are ubiquitous across the animal kingdom and exhibit ferroxidase activity, converting ferrous iron into a ferric form that is stably stored and soluble in the ferritin. Here, ferritin may serve as a source of biological iron for the worm through a process of iron acquisition, storage, and release during the light production process. This study addresses these options by assessing foundational data that characterize the ferroxidase activity of recombinant ChF with respect to human heavy‐chain ferritin (HuHF). ChF exhibits faster initial rates of iron oxidation than HuHF, but reaches an equilibrium state with detectable levels of ferrous iron still in solution; in contrast this was was not observed for HuHF that oxidizes all available iron in solution. This may support the hypothesis that ChF has a reducing activity. This could involve the release of ferric iron, which may be reduced by flavin molecules found in the mucus; the resulting ferrous iron could then subsequently undergo a Fenton reaction, acting as a source of electrons for long‐lasting mucus bioluminescence.
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