Proton Affinities of Some Amino Acid Side Chains in a Restricted Environment T. G. Abi, Amit Anand, and Srabani Taraphder* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India ReceiVed: September 29, 2008; ReVised Manuscript ReceiVed: April 16, 2009 We investigate the dependence of proton affinity values of the side chains of amino acids such as Asp, Glu, His, Ser, and Thr on confinement in a single-walled carbon nanotube. The proton affinity values, estimated using the density functional theories (PW91/dnp and BLYP/dnp), are found to be highly sensitive toward confinement. We find that for both Asp and Glu, the proton affinity, while suspended inside the carbon nanotube, becomes much less in comparison to their respective gas phase values. In the case of His, Ser, and Thr side chains, on the other hand, the proton affinity inside the carbon nanotube becomes negative. Hydrogen bonding with neighboring polar groups is found to result in a marked increase in proton affinity inside the tube in all of the cases reported in this article. The increase is most remarkable in the case of His, Ser, and Thr side chains where the presence of polar neighboring groups within a hydrogen-bonding distance is found to augment the proton affinity value by more than 100 kcal mol -1 . I. Introduction Proton uptake or release by an amino acid residue in a protein constitutes one of the most commonly encountered processes in bioenzymatic catalyzes. 1 To understand the molecular mech- anism of such steps at a given pH, different theoretical and experimental investigations may be designed. For example, to propose the basic mechanistic steps, the protonation states of titrable amino acid residues are often derived based on their pK a values. 2 However, there may be cases where considerations based on pK a values alone may not be sufficient to describe the molecular mechanism. In a predominantly nonequilibrium situation, for instance, pK a measurements give inadequate data about the underlying transition events. Therefore, it may be fundamentally important to investigate the suitability of other reactivity parameters as viable alternatives to be used in such cases. An interesting yet complicated case study is presented by the isozymes of carbonic anhydrase (CA). In the highly efficient isozyme human carbonic anhydrase (HCA) II, the rate- determining step involves an intramolecular proton transfer between a zinc-bound water (hydroxide) molecule and His-64 (PDB code: 2CBA) 3 with a maximal value of the rate constant k B ) 800 ms -1 . 4 This key histidine residue is located in a narrow channel leading from the surface of the enzyme into its active site. It is characterized by a pK a value of 7.2 4 facilitating proton transfer both from and to His as required by the reversible nature of the catalysis. This residue is also found in the isozymes CA IV and VII at the same location and has been connected to the high catalytic turnovers of these isozymes as well. 5 A much lower efficiency of the isozyme HCA III (k B ) 3 ms -1 ) 4 has been attributed to the presence of a Lys residue (pK a ∼ 9) at the position 64 that is too basic at physiological pH to serve as an efficient proton shuttle. 6 However, mutation of Lys by His (K64H) in HCA III results in a marginal recovery of the catalytic rate with k B ) 20.0 ms -1 in spite of the His residue having a pK a of 7.5. A somewhat higher efficiency of the proton shuttle may be obtained if a Glu or Asp residue is inserted at the location 64 in HCA III resulting in k B ∼ 40.0 ms -1 where both of the residues exhibit pK a values equal to 6.4 and 5.7, respectively. 7 The observed variations in efficiency of the putative proton shuttle as discussed above thus appear to depend not only on the pK a values but also on various other factors. Rotation of residue 64 through the channel-like pore and a nonequilibrium coupling of the protonation state of the side chain to its fluctuating orientation may be some of the major contributors. 8-11 As already indicated, the presence of specific neighboring residues and internal water molecules may also play a crucial role. 4,8-11 However, the variation of reactivity of different residues such as His, Asp, and Glu at position 64 restricted inside a channel-like environment has not been emphasized so far. In this article, we explore proton affinity (PA) as a reactivity parameter that is not limited by equilibrium considerations and investigate its sensitivity to confinement in a structural pore similar to the ones found in CAs. Interestingly, PA, rather than pK a , was found to drive the nonequilibrium steps in the photocycle of unidirectional proton transport in bacteriorho- dopsin. 12 However, as a prelude to studying the reactivity parameter inside an actual protein, we present in this article the variation of PA in model channel-like pores that closely resemble the one discussed in connection with CAs. We have investigated the proton affinities of some of the polar side chains of amino acid residues only as they are supposed to take part in the proton relay across a protein as suggested by Onsager for a model of long-range biological proton transport. 13,14 The PA of a molecule is generally defined as the change in enthalpy associated with the addition of a proton to a molecule B in the reaction B + H + f BH + . This is an extensively studied reactivity parameter of naturally occurring amino acids. 15,16 Conventionally, PA is measured in the gas phase using a wide variety of modern mass spectrometric techniques 17 and kinetic methods. 18 Ab initio and density functional theory (DFT) 19,20 calculations have also been carried out in the gas phase. In general, the PA of a molecule may be estimated as 21 * To whom correspondence should be addressed. E-mail: srabani@ chem.iitkgp.ernet.in. J. Phys. Chem. B 2009, 113, 9570–9576 9570 10.1021/jp808606b CCC: $40.75  2009 American Chemical Society Published on Web 06/23/2009