The crystal structure of recombinant pea cytosolic ascorbate peroxidase has been refined to an R = 0.19 for data between 8.0 and 2.2 A resolution and magnitude of F > or = 2 sigma(magnitude of F). The refined model consists of four ascorbate peroxidase monomers consisting of 249 residues per monomer assembled into two homodimers, with one heme group per monomer. The ascorbate peroxidase model confirms that the pea cytosolic enzyme is a noncovalent homodimer held together by a series of ionic interactions arranged around the 2-fold noncrystallographic dimer axis. As expected from the high level of sequence identity (33%), the overall fold of the ascorbate peroxidase monomer closely resembles that of cytochrome c peroxidase. The average root mean square differences for 137 helical alpha-carbon atoms between the four ascorbate peroxidase monomers and cytochrome c peroxidase and for 249 topologically equivalent alpha-carbon atoms are 0.9 and 1.3 A, respectively. The active site structures are also the same, including the hydrogen-bonding interactions between the proximal His ligand, a buried Asp residue, and a Trp residue, whose indole ring is parallel to and in contact with the proximal His ligand just under the heme ring. This proximal Trp residue is thought to be the site of free radical formation in cytochrome c peroxidase compound I and is also essential for enzyme activity. The corresponding Trp in ascorbate peroxidase, Trp179, occupies exactly the same position. The most interesting, and possibly functionally important, difference between the two peroxidases is the presence of a cation binding site in ascorbate peroxidase located approximately 8 A from the alpha-carbon atom of Trp179.

Electron paramagnetic resonance (EPR) spectroscopy has been used to analyze the ascorbate peroxidase Fe3+ resting state and to compare the reaction product between the enzyme and H2O2, compound I, with that of cytochrome c peroxidase. Because ascorbate peroxidase has a Trp residue in the proximal heme pocket at the same location as the Trp191 compound I free radical in cytochrome c peroxidase [Patterson, W. R., & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], it was anticipated that ascorbate peroxidase compound I might also contain a Trp-centered radical. However, the ascorbate peroxidase compound I EPR spectrum is totally different from that of cytochrome c peroxidase. Immediately after the addition of H2O2, the 7.5 K EPR spectrum of ascorbate peroxidase compound I exhibits an axial resonance extending from g perpendicular = 3.27 to g parallel approximately 2 that disappears within 30 s, presumably due to endogenous reduction of compound I. In contrast, cytochrome c peroxidase compound I exhibits a long-lived g approximately 2 signal associated with the Trp191 cation free-radical [Houseman, A. L. P., et al. (1993) Biochemistry 32, 4430-4443]. Recently, the 2 K EPR spectrum of a catalase compound I was found to exhibit a broad signal extending from g perpendicular = 3.45 to g parallel approximately 2 and was interpreted as a porphyrin pi cation radical [Benecky, M. J., et al. (1993) Biochemistry 32, 11929-11933]. On the basis of these comparisons, we conclude that ascorbate peroxidase forms an unstable compound I porphyrin pi cation radical, even though it has a Trp residue positioned precisely where the Trp191 radical is located in cytochrome c peroxidase.

The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.

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We have designed, fabricated, and characterized a microminiaturized "neuroport" for brain implantable neuroprosthesis applications, using an analog CMOS integrated circuit and a silicon based microelectrode array. An ultra-low power, low-noise CMOS preamplifier array with integral multiplexing was designed to accommodate stringent thermal and electrophysiological requirements for implantation in the brain, and a hybrid integration approach was developed to fabricate a functional microminiaturized neuroprobe device. Measurements showed that our fully scalable 16-channel CMOS amplifier chip had an average gain of 44 dB, bandwidth from 10 Hz to 7.3 kHz, and an equivalent input noise of approximately 9 microVrms with an average power consumption per preamplifier of 52 microW, which is consistent with simulation results. As a proof-of-concept demonstration, we have measured local field potentials from thalamocortical brain slices of rats, showing oscillatory behavior with an amplitude about 0.5 mV and a period ranging 80-120 ms. The results suggest that the hybrid integrated neuroport can form a prime platform for the development of a next level microminiaturized neural interface to the brain in a single implantable unit.