Synchrotron X-ray-Induced Photoreduction of Ferric Myoglobin Nitrite Crystals Gives the Ferrous Derivative with Retention of the O-Bonded Nitrite Ligand

Abstract

Exposure of a single crystal of the nitrite adduct of ferric myoglobin (Mb) at 100 K to highintensity synchrotron X-ray radiation resulted in changes in the UV-vis spectrum that can be attributed to reduction of the ferric compound to the ferrous derivative. We employed correlated single-crystal spectroscopy with crystallography to further characterize this photoproduct. The 1.55 Å resolution crystal structure of the photoproduct reveals retention of the O-binding mode of nitrite to the iron center. The data are consistent with the cryogenic generation and trapping, at 100 K, of a ferrous d 6 Mb II (ONO)* complex by photoreduction of the ferric precursor crystals using high-intensity X-ray radiation.Some mammalian proteins such as the heme-containing myoglobin (Mb) can reduce the nitrite anion (NO 2 − ; pK a 3.2) under hypoxic conditions to nitric oxide (NO; eq 1) (1,2) in a process somewhat reminiscent of that employed by denitrifying nitrite reductase (NiR) enzymes.(1)Although this subject area is under active investigation and debate, Gladwin and coworkers have demonstrated that nitrite protects against myocardial infarction in Mb +/+ mice, but does not in Mb −/− knockout mice, thus indeed implicating Mb as an in vivo NiR (1).Clearly, the ability of heme to facilitate nitrite reduction (eq 1) necessitates its presence in the ferrous form that can supply the electron needed for this process. We reported the X-ray crystal structure of the related stable ferric d 5 Mb-nitrite compound, and showed that the † This work was supported by the Oklahoma Center for the Advancement of Science and Technology (HR9-081, to GBR-A) and by SUPPORTING INFORMATION AVAILABLE X-ray data collection, structure solution and refinement; Movie in avi format showing the single-crystal spectra before and after highintensity X-ray exposure (for the process described in Fig. 4) as a function of crystal rotation angle. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 July 27. (4), and this nitro mode was observed in the X-ray crystal structures of the nitrite adducts of cyt cd 1 NiR (P. pantotrophus (5)), sulfite reductase hemoprotein (E. coli (6)), and cyt c NiR (W. succinogenes (7) and T. nitratireducens (8)). In contrast, the nitrito mode was observed in the nitrite adduct of ferric human hemoglobin (9).We were unable, however, and despite several varied attempts, to generate the physiologically important ferrous d 6 wild-type Mb-nitrite derivative to determine the nitrite binding mode (3). We have been intrigued by the use of synchrotron X-ray radiation for the generation of electrons in protein crystals (10). We have thus taken advantage of correlated microspectrophotometry and synchrotron X-ray photoreduction techniques to generate and characterize a ferrous d 6 Mb-nitrite compound at 100 K.Single crystals of ferric horse heart Mb III (ONO) at pH 7.4 were generated as previously described (3). The single...

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

“…Changes of similar magnitude have been inferred from XPR studies of other metalloproteins and synthetic complexes (53,54,58,60,61,(92)(93)(94). XPR even has been suggested as a tool for studying reduced states of metal cofactors (52,53,56,57).…”

Influence of iron doping on tetravalent nickel content in catalytic oxygen evolving films

“…4 have been obtained under OER conditions with less Fe incorporation. If X-rayinduced photoreduction were to give rise to this <1-eV change in edge energy (50,51), we would expect a systematic, instantaneous photoreduction across the different films. However, spectral changes do not change over multiple scans, suggesting that photoreduction of the film is not occurring upon continuous X-ray exposure.…”

A close look at dose: Toward L-edge XAS spectral uniformity, dose quantification and prediction of metal ion photoreduction

“…When performing XAS in the hard X-ray range (>2 keV), the energy (E) range of protein crystallography, one can for example study the metal K-edges of transition metal containing compounds. Here it is not the fading of diffraction spots, but the photoreduction of metal ions that is often readily observed, for example in references [53][54][55][56][57][58][59][60][61][62][63]. It is not our intention here to review the literature on metal K-edge XAS acquisition induced photoreduction, but it is important to note that metal photoreduction has been reported to occur below (1-2 orders of magnitude is not uncommon) crystallography's Henderson limit [59,61].…”