The positions of H atoms in macromolecules are never guaranteed using X-ray crystallographic techniques, even when atomic resolution (≤1.0 Å) can be obtained. If atomic resolution can be achieved, the interpretation of electron-density maps can still be ambiguous, especially when assigning the positions of H atoms of water molecules with high (>20 Å2
) thermal parameters (Gutberlet et al.
). However, neutron crystallographic data at medium (~2.0 Å) resolution can complement medium- to high-resolution (~2.0–1.0 Å) X-ray crystallographic data and allow the placement and analysis of key H atoms (Katz et al.
; Bennett et al.
; Fisher, Anderson et al.
; Blakeley, Ruiz et al.
; Coates et al.
; Blum et al.
H has a neutron scattering length (−3.7 × 10−15
m or −3.7 fm) that is similar in magnitude but opposite in sign to those of other atoms found in proteins (O, 5.8 fm; N, 9.4 fm; C, 6.6 fm; S, 2.8 fm), while deuterium (D) has a positive scattering length (6.7 fm) and a significantly smaller incoherent neutron-scattering cross-section (~2.0 barns versus
80 barns for H). Consequently, the signal-to-noise ratio can be markedly improved in neutron scattering when D atoms can be substituted for H atoms. In contrast to neutrons, the diffraction of X-rays depends on the number of electrons; as H and D are relatively electron-poor compared with the heavier atoms found in proteins, they diffract very weakly and appear practically invisible. All these factors together make it much easier to locate D or H in resulting nuclear density maps (Shu et al.
) and neutron crystallography can readily provide information on the protonation states of amino-acid residues, ligands and the nature of bonds involving hydrogen (Blakeley, Langan et al.
). Neutron crystallography can also be used to identify H atoms that are exchanged with D and the extent of this replacement, thus providing a tool for identifying isotopically labeled structural features, for studying solvent accessibility and macromolecular dynamics and for identifying minimal protein-folding domains (Bennett et al.
Neutron crystallography is also a powerful tool for studying the hydration of macromolecules, especially when it is combined with X-ray crystallography in joint (XN) refinement procedures (Blum et al.
). In electron-density maps, a water molecule is usually represented as a spherical density peak corresponding to the position of the O atom. However, in neutron scattering density maps, owing to the strong scattering contribution from D, the density associated with D2
O may no longer be spherical but rather extended. It can often be difficult to interpret these extended neutron scattering density peaks. However, we have found that using both X-ray and neutron data together can greatly help in this interpretation by allowing placement of the O atom and the subsequent interpretation of the extended neutron scattering density peak as either one or two D atoms. For this kind of analysis it is ideal to have neutron data to 2.0 Å resolution or better. Both the ability to accurately place and orient D2
O and to view the resulting hydrogen-bonded patterns they participate in can give enormous advantages in understanding how enzymes work.
Carbonic anhydrases (CAs) are ubiquitous enzymes that are found in all phyla of life and are intricately involved in many physiological processes (Tufts et al.
). Of all the isoforms, human carbonic anhydrase II (HCA II) is the most studied and probably the best understood. CAs catalyze the reversible hydration/dehydration of CO2
. The reaction occurs in two distinct steps: the conversion of CO2
and a subsequent proton-transfer (PT) step to regenerate the active site (see equations below; E
= enzyme, B
acceptor/donor, which can be buffer, His64 or bulk solvent; Christianson & Fierke, 1996
; Silverman & Lindskog, 1988
The first step is fairly well understood and a crystal structure of the enzyme–substrate complex was recently determined showing the substrate-binding pocket for the first time (Domsic et al.
; PDB code 3d92
). The second step, which is also the rate-limiting step of catalysis, is less well defined but is believed to proceed with the transfer of a H+
from the Zn-bound water to a proton-shuttling residue, His64, via
a hydrogen-bonded network of water molecules that span the 8 Å distance to the bulk solvent (Lindskog & Silverman, 2000
; Cui & Karplus, 2003
; Fisher et al.
; Fisher, Maupin et al.
). His64 sits on the edge of the cone-shaped active site and is ~8 Å away from the ZnOH−
O. In the crystal structures of HCA II determined at various pH values it has been observed that this proton shuttle can occupy two distinct conformations. The two conformations are the so-called ‘in’ and ‘out’ positions and it has been suggested that flexibility is a requirement for efficient PT from the solvent network to the bulk solvent (Nair & Christianson, 1991
; Fisher et al.
; Maupin & Voth, 2007
; Silverman & McKenna, 2007
). There are several important residues (Tyr7, Asn62, Asn67, Thr199 and Thr200) that line the active site that participate in hydrogen bonds with these water molecules and it is thought that they are important for ordering the network (Fisher, Tu et al.
Despite the abundance of kinetic and structural information on HCA II, the PT events in the catalytic cycle are not well understood or characterized. Even the determination of high-resolution X-ray crystal structures has failed to reveal the hydrogen-bonded details of the water network and the protonation states of the residues involved in the PT process (Duda et al.
; Fisher, Maupin et al.
). This enzyme provides a model system for the structure–function analyses of the role of hydrogen-bonded chains in rapid intramolecular PT over considerable distances in protein environments. HCA II and other CAs from diverse sources, overexpressed in bacterial systems, are also being widely developed for biomimetic carbon sequestration. CAs can be cheaply produced, are easily immobilized and work at a very high rate over a broad pH range, but their optimization for industrial application in carbon sequestration will require a detailed understanding of their catalytic cycle and the rate-limiting PT. Combining neutron crystallography with X-ray crystallography provides an ideal technique for observing the hydrogen-bonding networks and understanding their relation to PT in these and similar systems. To this end, H/D-exchanged crystals of HCA II have been prepared and X-ray and neutron crystallographic data have been collected at room temperature (RT) for joint XN crystallographic refinement of the water structure. Here, we report the preliminary data and feasibility of such a study.