The Influence of X‐Rays on the Structural Studies of Peroxide‐Derived Myoglobin Intermediates

Abstract

In recent years, the awareness of potential radiation damage of metal centers in protein crystals during crystallographic data collection has received increasing attention. The radiation damage can lead to radiation-induced changes and reduction of the metal sites. One of the research fields where these concerns have been comprehensively addressed is the study of the reaction intermediates of the heme peroxidase and oxygenase reaction cycles. For both the resting states and the high-valent intermediates, the X-rays used in the structure determination have given undesired side effects through radiation-induced changes to the trapped intermediates. However, X-rays have been used to generate and trap the peroxy/hydroperoxy state in crystals. In this review, the structural work and the influence of X-rays on these intermediates in myoglobin are summarized and viewed in light of analogous studies on similar intermediates in peroxidases and oxygenases.

Structures of K42N and K42Y sperm whale myoglobins point to an inhibitory role of distal water in peroxidase activity

“…Thus, the kinetic data and the spectroscopic data showing the formation of compound I by the mutant (Matsui et al, 1999) all indicate that ferric F43H/H64L binds H 2 O 2 much better than ferric Mb, while ferrous F43H/ H64L binds dioxygen much more poorly than ferrous Mb. For Mb, the binding geometries of O 2 and H 2 O 2 are almost identical (Hersleth et al, 2008); it may be expected that for F43H/H64L the geometry of O 2 binding is similar to that of H 2 O 2 binding. If so, it is likely that part of the differences in ligand binding to ferric F43H/H64L versus ferric Mb can be attributed to the lack of direct interaction of the distal histidine with the water molecule coordinated to the Fe in F43H/ H64L and the consequent weaker distal water stabilization.…”

“…3. The heme may be reduced and in the metastable aquo ferrous state (Hersleth et al, 2008), but for our comparative analysis of peroxidase function this probably has only a small effect since all of the structures are likely to be affected in a similar way. The individual structures of this region are shown in Supplementary Figs.…”

“…The distances at the heme in K42N and K42Y are compared with those in Mb in Table 2. It has been proposed, as reviewed recently by Hersleth et al (2008), that the major factor causing lower rates of peroxidase activity in Mb than in classical peroxidases is the lack of two residues: the Asp that forms the hydrogen bond to the proximal His (Sono et al, 1996) and the distal Arg that stabilizes the negative charge on the leaving group (Poulos & Kraut, 1980). Also, the shorter distance between the distal His and the heme Fe in Mb was considered to affect acid-base catalysis, leading to the heterolytic cleavage of hydrogen peroxide (Matsui et al, 1999).…”

Structural biology of redox partner interactions in P450cam monooxygenase: A fresh look at an old system

“…Here we face a serious conundrum since it has become increasingly clear that x-rays generate hydrated electrons that rapidly reduce redox centers in the proteins even at cryogenic temperatures [52]. Nevertheless, using careful data collection protocols with single crystal spectroscopy it is possible to obtain structures of metalloproteins in a well defined redox state [50][53].…”

Radiation Damage of Myoglobin Crystals in Weak Stationary Electric and Magnetic Fields

“…Myoglobin is one of the most studied biological molecules, especially in regard to radiation damage using X- and γ-rays [1]. The consequences of exposure to high energy photons on protein crystals, are specific structural changes, which lead to changes in global crystallographic parameters (I/σ(I), R cryst , mosaicity, anomalous signal and lattice dimensions).…”