The undisputed role of His64 in proton transfer during catalysis by carbonic anhydrases in the α class has raised questions concerning the details of its mechanism. The highly conserved residues Tyr7, Asn62, and Asn67 in the active-site cavity function to fine tune the properties of proton transfer by human carbonic anhydrase II (HCA II). For example, hydrophobic residues at these positions favor an inward orientation of His64 and a low pK a for its imidazole side chain. It appears that the predominant manner in which this fine tuning is achieved in rate constants for proton transfer is through the difference in pK a between His64 and the zinc-bound solvent molecule. Other properties of the active-site cavity, such as inward and outward conformers of His64, appear associated with the change in ΔpK a ; however, there is no strong evidence to date that the inward and outward orientations of His64 are in themselves requirements for facile proton transfer in carbonic anhydrase. Keywordscarbonic anhydrase; proton transfer; carbon dioxide; bicarbonate; hydration An important advance in understanding catalysis by carbonic anhydrase came in the report in 1975 from Steiner, Jonsson, and Lindskog [1] who used hydrogen/deuterium isotope effects to deduce a rate-limiting, intramolecular proton transfer in the maximum velocity of catalysis by human carbonic anhydrase II (HCA II). They suggested that His64 was the likely shuttle group based on its position in the active site and its pK a near 7 that was consistent with the kinetic pK a of k cat . Direct evidence that this suggestion was correct came 14 years later; it was the reduction in maximal velocity by about a factor of 20 caused by the replacement of His64 in HCA II with Ala which cannot support proton transfer [2]. Moreover, maximal velocity catalyzed by the mutant H64A HCA II was rescued to levels close to that of the wild-type enzyme by imidazole buffer in solution [2].Because of its rate-limiting, intramolecular proton transfer in catalysis, carbonic anhydrase has become a model for the examination of long range proton transfers and the role of hydrogenbonded water networks [1,3]. This is a step apparently shared by the numerous carbonic anhydrases classified into genetically distinct classes, such as α, β, and γ [4,5], all of which carry out catalysis of CO 2 hydration in a two-step mechanism. The first stage in the hydration Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBiochim Biophys Acta. Author manuscript; available in PMC 2011 February 1. direction is the reaction of zinc-b...

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Variants of human carbonic anhydrase II (HCA II) with amino-acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and 18O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing the replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for proton transfer from His64 to the zinc-bound hydroxide in catalysis were 4 μs-1 and 9 μs-1 for Y7F and Y7F-N67Q, respectively, compared with a value of 0.8 μs-1 for wild-type HCA II. These higher values observed for Y7F and Y7F-N67Q HCA II could not be explained by differences in the values of the pKa of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with reduced branching in the networks of hydrogen-bonded water molecules between the proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5 – 1.6 Å resolution. Moreover, Y7F-N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and Nδ of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.

Membrane inlet mass spectrometry was used to observe nitric oxide in the well-studied reaction of nitrite with hemoglobin. The membrane inlet was submerged in the reaction solutions and measured NO in solution via its flux across a semipermeable membrane leading to the mass spectrometer detecting the mass-to-charge ratio m/z 30. This method measures NO directly in solution and is an alternate approach compared with methods that purge solutions to measure NO. Addition to deoxyHb(Fe II ) (near 38 µM heme concentration) of nitrite in a range of 80 µM to 16 mM showed no accumulation of either NO or N 2 O 3 on a physiologically relevant time scale with a sensitivity near 1 nM. The addition of nitrite to oxy-Hb(Fe II ) and met-Hb(Fe III ) did not accumulate free NO to appreciable extents. These observations show that for several minutes after mixing nitrite with hemoglogin, free NO does not accumulate to levels exceeding the equilibrium level of NO. The presence of cyanide ions did not alter the appearance of the data; however, the presence of 2 mM mercuric ions at the beginning of the experiment with deoxy-Hb(Fe II ) shortened the initial phase of NO accumulation and increased the maximal level of free, unbound NO by about twofold. These experiments appear consistent with no role of met-Hb(Fe III ) in the generation of NO and an increase in nitrite reductase activity caused by the presumed binding of mercuric to cysteine residues. These results raise questions about the ability of reduction of nitrite mediated by deoxy-Hb(Fe II ) to play a role in vasodilation.