X-ray-induced photoreduction of heme metal centers rapidly induces active-site perturbations in a protein-independent manner

Abstract

Since the advent of protein crystallography, atomic-level macromolecular structures have provided a basis to understand biological function. Enzymologists use detailed structural insights on ligand coordination, interatomic distances, and positioning of catalytic amino acids to rationalize the underlying electronic reaction mechanisms. Often the proteins in question catalyze redox reactions using metal cofactors that are explicitly intertwined with their function. In these cases, the exact nature of the coordination sphere and the oxidation state of the metal is of utmost importance. Unfortunately, the redox-active nature of metal cofactors makes them especially susceptible to photoreduction, meaning that information obtained by photoreducing X-ray sources about the environment of the cofactor is the least trustworthy part of the structure. In this work we directly compare the kinetics of photoreduction of six different heme protein crystal species by X-ray radiation. We show that a dose of ∼40 kilograys already yields 50% ferrous iron in a heme protein crystal. We also demonstrate that the kinetics of photoreduction are completely independent from variables unique to the different samples tested. The photoreduction-induced structural rearrangements around the metal cofactors have to be considered when biochemical data of ferric proteins are rationalized by constraints derived from crystal structures of reduced enzymes.

Keywords: X-ray crystallography; enzyme structure; heme; heme proteins; oxidation-reduction (redox); protein crystallization; protein crystallography; radiation damage.

Figure 1.

Illustration of the diversity of secondary, tertiary and quaternary structure elements of the investigated heme proteins. A, secondary structure elements. α helices are shown in blue, β sheets are in red, and loop regions are shown as black lines. N and C termini are shown as yellow boxes. B, crystal structures of the investigated proteins. KpDyP (orange) and CCld (pink) form dimeric structures with one heme b moiety (gold) per subunit and a dimeric or truncated α-β ferredoxin fold. NdCld (turquoise) and LmChdC (yellow) form pentamers with one heme b or coproheme moiety per subunit and an α/β ferredoxin fold. hhMb (green) is a monomeric α-helical protein with one heme b cofactor. AvTsdA (blue) is a monomeric protein with two heme c cofactors and a predominantly α-helical structure. The protein backbone and cofactors are depicted as cartoon and ball-and-stick representations, respectively.

Figure 2.

Representative crystal forms and sizes of the investigated heme proteins. A, crystal forms and sizes of KpDyP (top, left), NdCld (top, middle), CCld (top, right), hhMb (bottom, left), AvTsdA (bottom, middle), and LmChdC (bottom, right). B, visualized impact of X-ray–induced reduction as seen on a mounted crystal of LmChdC. Red dots (indicated by the orange arrows) show areas hit by the X-ray beam. C, 750-μm mesh loaded with crystals from KpDyP and close up. Mesh cryo-cooling allows the rapid manual collection of up to 30–50 crystals without the need for time-consuming sample changing.

Figure 3.

Online microspectroscopy. Overlay of single-crystal UV-visible spectra obtained from online microspectroscopy (10% transmission) of the ferric (gray), ∼50% reduced (lightly shaded), and final (fully reduced, dark shaded) crystals of KpDyP (orange), NdCld (turquoise), CCld (pink), hhMb (green), AvTsdA (blue), and LmChdC (yellow). Arrows indicate the distinct increase in absorbance at the wavelength that was used to monitor the progress of photoreduction.

Figure 4.

Kinetics of radiation-mediated reduction of Fe(III) to Fe(II). A, representative dose traces (at 5% transmission) with single-exponential fits to derive the rate of reduction (k_red). B, rates of reduction (k_red) for all proteins in the respective color and the calculated average for all six proteins (in red). C, representative dose-dependent changes of absorption at protein-specific wavelength (see Fig. 3) at 5% transmission (flux: 2.1 × 10¹¹ photons s⁻¹) with hyperbolic fits to derive the dose of half-maximal reduction. D, doses needed to reduce the heme iron in the protein crystals to obtain 50% ferric and ferrous heme (average value for all proteins in red). Dose traces in A and C were lifted by multiples of 0.3 for each sample for better visibility. Plotted values in B and D calculated using RADDOSE-3D version 4.0 with individual input for all different samples are shown in the respective color for each sample. Calculations using RADDOSE-3D version 4.0 input from a generic heme protein crystal are shown in gray beside the respective individual data points (KpDyP in orange, NdCld in turquoise, CCld in pink, hhMb in green, AvTsdA in blue, and LmChdC in yellow). Error bars, S.D.

Figure 5.

Kinetics of radiation-mediated reduction of Compound I to Fe(II) in KpDyP. Shown are time- and dose-dependent changes in absorption at 555 nm of the ferric resting state (orange) and the Compound I intermediate (green) of KpDyP upon photoreduction (10% transmission, flux = 4.2 × 10¹¹ photons s⁻¹). Inset (bottom), overlay of single-crystal UV-visible spectra of the ferric (gray) resting state and the Compound I intermediate (black) obtained by soaking with H₂O₂. Inset (middle), ferric resting state before (gray) and after (orange) exposure. Inset (top), Compound I intermediate before (black) and after (green) exposure.

Figure 6.

Structural changes upon reduction of ferric high-spin KpDyP. Asp-143 (sticks), distal active-site water molecules, and the heme iron (sphere) are represented from KpDyP subunit A (top) and B (bottom) for the 5% reduced (blue, 6RQY), the 100% reduced (gold, 6RR8) structure, based on UV-visible spectroscopy, from the multicrystal approach and for the completely reduced routine single-crystal data set (red, 6FKS). 2mF_o − DF_c (contoured at 1 σ) electron density maps for the represented molecules are shown in the respective colors on the right.

Figure 7.

Structural changes upon reduction of ferric cyanide bound low-spin KpDyP. A, heme b, Asp-143, and cyanide (sticks) are represented from KpDyP subunit A (top) and subunit B (bottom) for the 5% reduced (blue, gray mesh, 6RPE) structure derived from the multicrystal approach and for the completely reduced routine single-crystal data set (purple, pink mesh, 6RPD). 2mF_o − DF_c electron density maps are represented for all modeled structural elements (contoured at 1.5 σ (cyanide alone) or 1 σ (rest)). B, flexibility of Asp-143 in subunit A (top) and subunit B (bottom) in the same colors as in A, representing the same states of reduction.