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Exposure of a single crystal of the nitrite adduct of ferric myoglobin (Mb) at 100 K to high-intensity 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 d6 MbII(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 (NO2−; pKa 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.
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 d5 Mb-nitrite compound, and showed that the nitrite ligand adopted an O-binding mode (i.e., nitrito; Fig. 1) (3). A valid criticism of this reported ferric MbIII(ONO) structure is that it may not represent the true binding mode of nitrite to the ferrous Mb just prior to its reduction to NO in vivo. For example, nitrite adopts an N-binding mode in its complex with the ferric Mb H64V mutant (4), and this nitro mode was observed in the X-ray crystal structures of the nitrite adducts of cyt cd1 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 d6 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 d6 Mb-nitrite compound at 100 K.
Single crystals of ferric horse heart MbIII(ONO) at pH 7.4 were generated as previously described (3). The single-crystal UV-vis spectrum of ferric MbIII(ONO) at 100 K is shown in Fig. 2A. Exposure of the crystal to low-intensity X-rays (1.0 × 109 photons/s; ~3% of max. flux) at 100 K at the NSLS that essentially reproduced our “home source” flux (0.7 × 109 photons/s) for 95 mins (calc. total X-ray dose of ~0.046 MGy (11)) resulted in only a very slight change of the spectrum from that of the non-X-ray-exposed crystal (Fig. 2B). In contrast, exposure of the same crystal of ferric MbIII(ONO) to high-intensity X-rays (3 × 1010 photons/s; λ = 1.0 Å) at a different location along the long, narrow, thin plate morphology of the crystal for 50 min (calc. total X-ray dose ≈ 0.42 MGy) resulted in rapid and dramatic changes in the optical spectrum (Fig. 2C). In particular, the Soret band is red-shifted, and the shape of the bands in the 500–600 nm region undergoes a significant change with the appearance of a relatively intense band at 570 nm. We hypothesized that the changes observed in Fig. 2C (i.e., after exposure of the crystal to high-intensity X-rays) were due to the formation and cryo-trapping of a six-coordinate ferrous d6 Mb-nitrite derivative (eq 2; * represents X-ray photoreduced species). Our hypothesis
was based on the fact that the spectral changes observed during exposure of ferric MbIII(ONO) (Fig. 2C) to high-intensity X-rays at 100 K are very similar to those observed by Schlichting (10) and Parak (12) when ferric aquometMb undergoes X-ray photoreduction at cryogenic temperatures to generate the metastable ferrous MbII(H2O) (eq 3 and Supporting Information).
To the best of our knowledge, the UV-vis spectrum of authentic ferrous MbII(ONO) has not been reported. We note, however, that the UV-vis spectrum of the product generated during photoreduction of ferric MbIII(ONO) (eq 2) is also similar to that of the ferrous six-coordinate MbII(CN) containing an anionic sixth ligand (13).
We then turned to correlated spectroscopy-crystallography to identify the products from the exposure of crystals of ferric MbIII(ONO) to low- and high-intensity X-rays. The UV-vis spectrum of a crystal that had been exposed to “home source” X-ray radiation for data collection is shown on the left of Fig. 3; the 1.60 Å resolution crystal structure obtained from the diffraction data (flux 0.7 × 109 photons/s; 0.3 mm beam diameter; Table 1) is shown on the right. The spectrum confirms that no significant photoreduction occurred during our home-source X-ray data collection (c.f., Fig. 2B). Figure 4 (left) shows the optical spectra of a ferric MbIII(ONO) crystal before (solid line), during (dashed line; after image 161; calc. total X-ray dose ≈ 0.45 MGy), and after (grey line; after image 365; calc. total X-ray dose ≈ 1.0 MGy) exposure to high-intensity X-rays. Although we collected 365 diffraction image frames (each with 1° oscillation), we only used frames 161–365 for the structure determination because the optical spectra indicated that the product formed during the latter portion of the X-ray exposure was stable. The resulting 1.55 Å resolution crystal structure is shown at the right of Fig. 4. Importantly, the UV-vis spectral changes are consistent with photoreduction to the ferrous d6 MbII(ONO)* derivative, and the X-ray crystal structure reveals the retention of the O-bound nitrite ligand. This O-binding mode was also observed in a related cobalt-substituted d6 CoIIIMb(ONO) complex (14).
The successful generation of a ferrous MbII(ONO)* complex using high-intensity X-ray radiation may represent a pathway to a non-equilibrium structure in which the O-bonded nitrite is trapped in this position with limited movement in the distal pocket at 100 K. Thus, it may not necessarily represent the true equilibrium structure of the reactive ferrous MbII-nitrite compound in solution at physiological temperature. Indeed, a similar non-equilibrium retention of the water ligand in X-ray-photoreduced metastable MbII(H2O) was obtained at <100 K (12); the complex loses its water ligand at temperatures above 150 K to give the well-characterized five-coordinate deoxy-Mb form obtained by chemical reduction of ferric MbIII(H2O). Further, Liu et al. have noted differences in the X-ray crystal structures of Thermus cyt ba3 oxidase reduction products depending on whether X-ray photoreduction or chemical reduction was employed (15); the structure of the active site of the X-ray photoreduced enzyme at 100 K showed retention of the bridging water molecule at the Fe-Cu bimetallic active site, in contrast to the structure of the dithionite-reduced ba3 that did not show such a bridging water molecule.
In summary, we have successfully generated and cryotrapped a nitrite adduct of ferrous d6 MbII at 100 K. This represents the first reported crystal structure of a nitrite adduct of any ferrous mammalian heme protein. Experiments to investigate the thermal stability and reactivity of this MbII(ONO)* complex are underway.
Data for this study were measured, in part, at beamline X26-C of the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory. Use of the NSLS was supported by the U.S. Department of Energy Office of Basic Energy Sciences, under Contract DE-AC02-98CH10886. We thank Dr. Timothy Sage for alerting us to reference 12.
SUPPORTING INFORMATION AVAILABLE
X-ray data collection, structure solution and refinement; Movie in avi format showing the single-crystal spectra before and after high-intensity 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.
†This work was supported by the Oklahoma Center for the Advancement of Science and Technology (HR9-081, to GBR-A) and by the Office of Biological and Environmental Research, U.S. Department of Energy and the National Center for Research Resources (2 P41 RR012408, to AMO) of the National Institutes of Health.