Enzymes for carbon sequestration: neutron crystallographic studies of carbonic anhydrase.

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme that catalyzes the reversible hydration of CO(2) to form HCO(3)(-) and H(+) using a Zn-hydroxide mechanism. The first part of catalysis involves CO(2) hydration, while the second part deals with removing the excess proton that is formed during the first step. Proton transfer (PT) is thought to occur through a well ordered hydrogen-bonded network of waters that stretches from the metal center of CA to an internal proton shuttle, His64. These waters are oriented and ordered through a series of hydrogen-bonding interactions to hydrophilic residues that line the active site of CA. Neutron studies were conducted on wild-type human CA isoform II (HCA II) in order to better understand the nature and the orientation of the Zn-bound solvent (ZS), the charged state and conformation of His64, the hydrogen-bonding patterns and orientations of the water molecules that mediate PT and the ionization of hydrophilic residues in the active site that interact with the water network. Several interesting and unexpected features in the active site were observed which have implications for how PT proceeds in CA.

Preliminary joint neutron and X-ray crystallographic study of human carbonic anhydrase II.

Carbonic anhydrases catalyze the interconversion of CO(2) to HCO(3)(-), with a subsequent proton-transfer (PT) step. PT proceeds via a proposed hydrogen-bonded water network in the active-site cavity that is stabilized by several hydrophilic residues. A joint X-ray and neutron crystallographic study has been initiated to determine the specific water network and the protonation states of the hydrophilic residues that coordinate it in human carbonic anhydrase II. Time-of-flight neutron crystallographic data have been collected from a large ( approximately 1.2 mm(3)) hydrogen/deuterium-exchanged crystal to 2.4 A resolution and X-ray crystallographic data have been collected from a similar but smaller crystal to 1.5 A resolution. Obtaining good-quality neutron data will contribute to the understanding of the catalytic mechanisms that utilize water networks for PT in protein environments.

Preliminary time-of-flight neutron diffraction study of human deoxyhemoglobin.

Human hemoglobin (HbA) is an intricate system that has evolved to efficiently transport oxygen molecules (O(2)) from lung to tissue. Its quaternary structure can fluctuate between two conformations, T (tense or deoxy) and R (relaxed or oxy), which have low and high affinity for O(2), respectively. The binding of O(2) to the heme sites of HbA is regulated by protons and by inorganic anions. In order to investigate the role of the protonation states of protein residues in O(2) binding, large crystals of deoxy HbA (approximately 20 mm(3)) were grown in D(2)O under anaerobic conditions for neutron diffraction studies. A time-of-flight neutron data set was collected to 1.8 A resolution on the Protein Crystallography Station (PCS) at the spallation source run by Los Alamos Neutron Science Center (LANSCE). The HbA tetramer (64.6 kDa; 574 residues excluding the four heme groups) occupies the largest asymmetric unit (space group P2(1)) from which a high-resolution neutron data set has been collected to date.