Structure of a Zinc Porphyrin-Substituted Bacterioferritin and Photophysical Properties of Iron Reduction.

The iron storage protein bacterioferritin (Bfr) binds up to 12 hemes b at specific sites in its protein shell. The heme b can be substituted with the photosensitizer Zn(II)-protoporphyrin IX (ZnPP), and photosensitized reductive iron release from the ferric oxyhydroxide {[FeO(OH)]n} core inside the ZnPP-Bfr protein shell was demonstrated [Cioloboc, D., et al. (2018) Biomacromolecules 19, 178-187]. This report describes the X-ray crystal structure of ZnPP-Bfr and the effects of loaded iron on the photophysical properties of the ZnPP. The crystal structure of ZnPP-Bfr shows a unique six-coordinate zinc in the ZnPP with two axial methionine sulfur ligands. Steady state and transient ultraviolet-visible absorption and luminescence spectroscopies show that irradiation with light overlapping the Soret absorption causes oxidation of ZnPP to the cation radical ZnPP•+ only when the ZnPP-Bfr is loaded with [FeO(OH)]n. Femtosecond transient absorption spectroscopy shows that this photooxidation occurs from the singlet excited state (1ZnPP*) on the picosecond time scale and is consistent with two oxidizing populations of Fe3+, which do not appear to involve the ferroxidase center iron. We propose that [FeO(OH)]n clusters at or near the inner surface of the protein shell are responsible for ZnPP photooxidation. Hopping of the photoinjected electrons through the [FeO(OH)]n would effectively cause migration of Fe2+ through the inner cavity to pores where it exits the protein. Reductive iron mobilization is presumed to be a physiological function of Bfrs. The phototriggered Fe3+ reduction could be used to identify the sites of iron mobilization within the Bfr protein shell.

Structural, Photophysical, and Photochemical Characterization of Zinc Protoporphyrin IX in a Dimeric Variant of an Iron Storage Protein: Insights into the Mechanism of Photosensitized H2 Generation.

Some of us have previously reported the preparation of a dimeric form of the iron storage protein, bacterioferritin (Bfr), in which the native heme b is substituted with the photosensitizer, Zn(II)-protoporphyrin IX (ZnPP-Bfr dimer). We further showed that the ZnPP-Bfr dimer can serve as a photosensitizer for platinum-catalyzed H2 generation in aqueous solution without the usually added electron relay between photosensitizer and platinum ( Clark , E. R. , Inorg. Chem. 2017 , 56 , 4584 - 4593 ). We proposed reductive or oxidative quenching pathways involving the ZnPP anion radical (ZnPP•-) or the ZnPP cation radical, (ZnPP•+), respectively. The present report describes structural, photophysical, and photochemical properties of the ZnPP in the ZnPP-Bfr dimer. X-ray absorption spectroscopic studies at 10 K showed a mixture of five- and six-coordinated Zn centers with axial coordination by one long Zn-SγMet distance of ∼2.8 Å and ∼40% having an additional shorter Zn-S distance of ∼2.4 Å, in addition to the expected 4 nitrogen atom coordination from the porphyrin. The ZnPP in ZnPP-Bfr dimer was prone to photosensitized oxidation to ZnPP•+. The ZnPP•+ was rapidly reduced by ascorbic acid, which we previously determined was essential for photosensitized H2 production in this system. These results are consistent with an oxidative quenching pathway involving electron transfer from 3ZnPP* to platinum, which may be assisted by a flexible ZnPP axial coordination sphere. However, the low quantum yield for H2 production (∼1%) in this system could make reductive quenching difficult to detect, and can, therefore, not be completely ruled out. The ZnPP-Bfr dimer provides a simple but versatile framework for mechanistic assessment and optimization of porphyrin-photosensitized H2 generation without an electron relay between porphyrin and the platinum catalyst.

Thermal Treatment Greatly Improves Storage Stability and Monodispersity of Pea Seed Ferritin.

Plant ferritin in holo form is considered as a novel, ideal iron supplement for human nutrition in the 21st century, but its self-degradation and self-association features limit its application on account of the presence of extension peptide (EP), a specific domain only found in plant ferritin. Although reported chemicals such as Phenylmethanesulfonyl fluoride (PMSF) can inhibit its self-degradation, they are not edible and toxic. In the present work, we found that thermal treatment of pea seed ferritin (PSF) in the range of 60 to 80 °C can prolong the storage time of PSF from 3 days to at least 10 days. In the meanwhile, the aggregated form can be inhibited upon such treatment, therefore promoting its monodispersity. More important, such treatment had little effect on the natural shell-like structure of holo PSF and its iron content. In contrast, thermal treatment at higher temperature (90 °C or above) resulted in a change in ferritin structure. These new findings pave the way to the application of plant ferritin as an iron supplement. PRACTICAL APPLICATION: Thermal treatment at 60 to 80 °C can prolong the storage stability of PSF from 3 days to at least 10 days and prevent it from self-aggregation without affecting the shell-like structure. It has been known that the stability of PSF is closely associated with the bioavailability of iron within PSF. From the standpoint of nutrition, the above-mentioned thermal treatment could be used as a cooking method in our daily life or in food industry to improve the bioavailability of ferritin iron, thereby being beneficial for exploration of plant ferritin as a novel, ideal iron supplement to fight against IDA.

High-resolution electron microscopy and spectroscopy of ferritin in biocompatible graphene liquid cells and graphene sandwiches.

Atomic and electronic structures of hydrated ferritin are characterized using electron microscopy and spectroscopy through encapsulation in single layer graphene in a biocompatible manner. Graphene's ability to reduce radiation damage levels to hydrogen bond breakage is demonstrated. A reduction of iron valence from 3+ to 2+ is measured at nanometer-resolution in ferritin, showing initial stages of iron release by ferritin.

Reduction of X-ray-induced radiation damage of macromolecular crystals by data collection at 15 K: a systematic study.

The cryocooling of protein crystals to temperatures of around 100 K drastically reduces X-ray-induced radiation damage. The majority of macromolecular data collection is therefore performed at 100 K, yielding diffraction data of higher resolution and allowing structure determination from much smaller crystals. However, at third-generation synchrotron sources radiation damage at 100 K still limits the useful data obtainable from a crystal. For data collection at 15 K, realised by the use of an open-flow helium cryostat, a further reduction of radiation damage is expected. However, no systematic studies have been undertaken so far. In this present study, a total of 54 data sets have been collected from holoferritin and insulin crystals at 15 and 90 K in order to identify the effect of the lower data-collection temperature on the radiation damage. It is shown that data collection at 15 K has only a small positive effect for insulin crystals, whereas for holoferritin crystals radiation damage is reduced by 23% compared with data collection at 90 K.

Experimental determination of the radiation dose limit for cryocooled protein crystals.

Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular diffraction data collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 x 10(7) Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program raddose after measurement of the incident photon flux and determination of the elemental crystal composition by micro-particle-induced x-ray emission. Degradation in diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose/dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half (D(1/2)). From these measurements, a dose limit of D(1/2) = 4.3 (+/-0.3) x10(7) Gy was obtained. However, by considering other data quality indicators, an intensity reduction to I(ln2) = ln2 x I(0), corresponding to an absorbed dose of 3.0 x 10(7) Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.

Radiation driven collapse of protein crystals.

During coherent X-ray diffraction measurements on crystals of ferritin at room temperature using monochromatic undulator radiation from the Advanced Photon Source, a sudden lattice contraction was observed following a characteristic latent period and ultimately leading to the collapse of the crystal. The progression of this collapse is analysed using a two-state Hendricks-Teller model. It reveals that 55% of the layers collapse by 1.6% before the crystal completely stops diffracting.

Iron release analyses from ferritin by visible light irradiation.

We investigated the iron release from ferritin by irradiation from a white fluorescent light in the absence or presence of ADP. Irradiation of a ferritin solution at 17,000 lx in the absence of ADP slightly induces iron release from ferritin but only at acidic pH conditions (pH 5.0 or pH 6.0). Irradiation in the presence of ADP markedly enhances iron release from ferritin under the same conditions. In the absence of irradiation, the iron release from ferritin was low even in the presence of ADP. The induction of the iron release by irradiation in the presence of ADP was also affected by various factors such as irradiation dose and acidity, but not temperature (4-47 degrees C), oxygen concentration, or free radical generations during the irradiation. The iron release during the irradiation ceased to increase by turning off the light and was found to increase again after additional irradiation. These results suggest that visible light directly induces iron release from ferritin via the photoreduction of iron stored inside ferritin.

Different onsets of oxidative damage to DNA and lipids in bone marrow and liver in rats given total body irradiation.

We examined time-dependent changes in antioxidant vitamins and oxidative damage to DNA and lipids in the bone marrow, liver, and plasma of rats given total body irradiation (TBI) with X-rays at 3 Gy. The oxidative damage to DNA and lipids was evaluated by measuring increases of 8-hydroxydeoxyguanosine (8OHdG) in DNA and 4-hydroxy-2-nonenal (HNE), respectively. After the TBI, marked increases in 8OHdG and HNE were detected at 3 to 5 h in the bone marrow, while gradual increases in these parameters were detected after a few days in the liver. These changes in 8OHdG and HNE were well correlated within each tissue. In the bone marrow, levels of both vitamin C and vitamin E were decreased by the TBI; however, the changes in vitamin C were earlier and greater than those in vitamin E. In the liver, the level of vitamin C did not decrease, but that of vitamin E decreased due to the TBI. Changes in HNE, vitamin C, and vitamin E in the plasma were similar to those in the liver. Within each tissue, the time of decrease in antioxidants was almost the same as that of the increase in oxidative damage. An increase in total iron due to the TBI was also detected in these tissues. In particular, the total iron in the bone marrow was markedly increased at a few hours after the TBI, with a slight increase in transferrin and no increase in ferritin. Exposure studies performed on cells or isolated DNA showed that an increase in 8OHdG was detected immediately after irradiation at more than 100 Gy in bone marrow cells and at less than 10 Gy in isolated DNA, suggesting that an increase in 8OHdG is undetectable even in bone marrow immediately after the TBI at 3 Gy. These results indicate that the onset of oxidative damage to DNA and lipids was delayed after TBI at 3 Gy, that it was quite different in the bone marrow and the liver, and that an increase in iron and decrease in antioxidant vitamins were involved in the mechanism of oxidative damage.

Oxidative defense in cultured human skin fibroblasts and keratinocytes from sun-exposed and non-exposed skin.

Skin fibroblasts and keratinocytes cultivated from chronically light-exposed skin sites have higher levels of the protective protein ferritin than cells derived from unexposed areas of the body, suggesting an adaptive response of cells exposed to chronic external insults. In the same line, ferritin levels were always found to be 2- to 7-fold higher in epidermal keratinocytes than in the underlying dermal fibroblasts of the same person thus providing the keratinocyte with continuous protection by the higher cellular ferritin content. The activation of ferritin by oxidative stress including UVA radiation could represent an important cellular defense mechanism that operates in human skin. Following low fluences of UVA radiation (2-4 x 10(5) J/m2), ferritin levels increased by 20-30% in normal adult skin fibroblasts and showed a subsequent decrease at higher UVA fluences. In contrast, skin keratinocytes were not perturbed by UVA radiation exposure except for very high fluences (1.25 x 10(6) J/m2) where slight decreases in cellular ferritin were noted in 7 of the 12 cell lines. Fibroblasts derived from light-exposed skin sites that possessed higher levels of cellular ferritin were highly protected against UVA-induced membrane damage as measured by lactate dehydrogenase release compared with fibroblasts from nonexposed body sites with lower levels of ferritin. It is clear from our results that ferritin plays an important role in protection at the cellular level in human skin cells, but the role of this putative protective protein in vivo remains to be defined.

Peroxidation of model lipoprotein solutions sensitized by photoreduction of ferritin by 365 nm radiation.

A mechanistic study involving the 365 nm irradiation of aerated, phosphate-buffered solutions of human high-density lipoproteins (HDL3 fraction) and ferritin was undertaken. The 365 nm irradiation of phosphate-buffered horse spleen ferritin solutions induces the release of Fe2+ in the medium. The initial quantum yield of Fe2+ release on irradiation is 0.002. This quantum yield is oxygen independent. The 365 nm irradiation of mixtures of HDL and ferritin leads to alterations in apolipoproteins as revealed by tryptophan (Trp) oxidation and electrophoretic pattern modification. In parallel with protein damage, lipid peroxidation is induced as shown by hydroperoxide and thiobarbituric acid reactive substances (TBARS) formation. These peroxidations are strongly reduced in 0.1 M formate solution, which suggests chain initiation by .OH radicals or subsequent radicals produced by .OH. They are completely inhibited by desferrioxamine, consistent with propagation by Fe2+ ion. By contrast incubation of HDL in the presence of ferritin and FeSO4 induces only poor auto-oxidation. The biological relevance of this study is discussed.

Ferrous ion release from ferritin by ultraviolet-A radiations.

Ferritin is the principal protein of iron storage (in the Fe(III) state). The UV-A irradiation of 0.25 microM ferritin solutions (from horse spleen) loaded with 530 microM Fe(III) induces Fe2+ release in the medium. The initial quantum yield is wavelength dependent (phi(365 nm) approximately 2 x 10(-3) but pH and oxygen independent. The Fe2+ release reaches a plateau which strongly depends on pH and oxygen. The amino acid composition of the apoprotein is not altered by the UV irradiation. Addition of formate ions enhances the Fe2+ production, suggesting that the ferritin photoreduction involves an electron transfer from an OH- ligand. The possible importance of this phenomenon in skin photobiology is discussed.