Crystallographic and Single-Crystal Spectral Analysis of the Peroxidase Ferryl Intermediate

Meharenna, Yergalem T.; Doukov, Tzanko; Li, Huiying; Soltis, S. Michael; Poulos, T.L.

doi:10.1021/bi100238r

Cited by 76 publications

(108 citation statements)

References 14 publications

Supporting

Mentioning

104

Contrasting

Order By: Relevance

“…For CcP Compound I, the EXAFS and most of the other data now seem to support an unprotonated ferryl-oxy species (32,35,36); this is in agreement with our analyses for both CcP Compound I and APX Compound I. Compounds I of HRP (8,(37)(38)(39) and chloroperoxidase (39) are also not protonated.…”

Section: Discussionsupporting

confidence: 89%

Nature of the Ferryl Heme in Compounds I and II

Gumiero

Metcalfe

Pearson

et al. 2011

Journal of Biological Chemistry

105

View full text Add to dashboard Cite

Section: Discussionsupporting

confidence: 89%

Nature of the Ferryl Heme in Compounds I and II

Gumiero

Metcalfe

Pearson

et al. 2011

Journal of Biological Chemistry

105

View full text Add to dashboard Cite

“…Kβ 1,3 and Kβ′ lines provide intrinsic information about the electronic structure and the advancement of the catalytic intermediates. XES can also serve as an in situ method for monitoring the integrity of metal catalytic centers during XRD data collection; it has been shown in previous SR work (29)(30)(31)(32) that an active site can already be fully damaged when the overall protein crystal structure is still intact.…”

Section: Resultsmentioning

confidence: 99%

“…This is in particular the case for most biological and biomimetic systems that work in dilute, aqueous environments. Therefore, at SR sources, X-ray absorption and emission spectra of most redox active biological/aqueous systems are collected under cryogenic conditions, thus minimizing diffusion of hydroxyl and other radical species that are the major cause of radiation damage (29)(30)(31)(32).…”

mentioning

confidence: 99%

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode

Alonso-Mori

Kern

Gildea

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

125

View full text Add to dashboard Cite

The ultrabright femtosecond X-ray pulses provided by X-ray freeelectron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this "probe-before-destroy" approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. Kβ 1,3 XES spectra of Mn II and Mn 2 III,IV complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to >100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shotby-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.energy-dispersive XES | Kβ emission lines | femtosecond x-ray spectroscopy T he first X-ray free-electron laser (XFEL) operating in the hard X-ray regime (1), the Linac Coherent Light Source (LCLS), produces ∼5-to 400-fs X-ray pulses with up to ∼10 12 photons per pulse at 6-10 keV at a repetition rate of 120 Hz. Each of these X-ray pulses is intense enough to expel multiple electrons from the target, which can lead to a Coulomb explosion that destroys the sample. In a shot-by-shot experiment, data are collected from each pulse before the destruction of the sample, and the sample is replenished after each pulse. The feasibility of this "probebefore-destroy" approach for X-ray crystallography experiments using XFEL pulses was first demonstrated by Chapman et al. with various systems and has subsequently been corroborated by others at the LCLS (2-6).Whereas X-ray crystallography is an important method for studying the geometric structure of entire complexes, X-ray absorption and emission spectroscopy are powerful techniques for studying the local chemistry in both inorganic systems and metalloproteins. X-ray absorption spectroscopy (XAS), where the unoccupied states are probed, has long been applied to various systems using synchrotron radiation (SR) (7-9), and recent advances in this methodology have made it possible to conduc...

show abstract

“…Based on our previous experience with yeast CCP (24), it is likely that LmP-LmCytc complex is a mix of redox states. Given that Cytc exhibits a higher redox potential, the LmCytc heme iron should be more susceptible to photoreduction, although the reported differences between oxidized and reduced Cytc (25) are subtle so we cannot conclude which redox state dominates based on such parameters as ligand-iron distances.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the Leishmania major peroxidase–cytochrome c complex

Jasion

Doukov

Pineda

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The causative agent of leishmaniasis is the protozoan parasite Leishmania major . Part of the host protective mechanism is the production of reactive oxygen species including hydrogen peroxide. In response, L. major produces a peroxidase, L. major peroxidase (LmP), that helps to protect the parasite from oxidative stress. LmP is a heme peroxidase that catalyzes the peroxidation of mitochondrial cytochrome c . We have determined the crystal structure of LmP in a complex with its substrate, L. major cytochrome c (LmCytc) to 1.84 Å, and compared the structure to its close homolog, the yeast cytochrome c peroxidase–cytochrome c complex. The binding interface between LmP and LmCytc has one strong and one weak ionic interaction that the yeast system lacks. The differences between the steady-state kinetics correlate well with the Lm redox pair being more dependent on ionic interactions, whereas the yeast redox pair depends more on nonpolar interactions. Mutagenesis studies confirm that the ion pairs at the intermolecular interface are important to both k cat and K M . Despite these differences, the electron transfer path, with respect to the distance between hemes, along the polypeptide chain is exactly the same in both redox systems. A potentially important difference, however, is the side chains involved. LmP has more polar groups (Asp and His) along the pathway compared with the nonpolar groups (Leu and Ala) in the yeast system, and as a result, the electrostatic environment along the presumed electron transfer path is substantially different.

show abstract

Crystallographic and Single-Crystal Spectral Analysis of the Peroxidase Ferryl Intermediate

Cited by 76 publications

References 14 publications

Nature of the Ferryl Heme in Compounds I and II

Nature of the Ferryl Heme in Compounds I and II

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode

Crystal structure of the Leishmania major peroxidase–cytochrome c complex

Contact Info

Product

Resources

About