Peters, John | Engineering Papers

DOE OSTI2025

Long-Range Allosteric Communication Modulated by Active Site Mn(II) Coordination Drives Catalysis in Xanthobacter autotrophicus Acetone Carboxylase

Acetone carboxylase (AC) from Xanthobacter autotrophicus is a 360 KDa α2β2γ2 heterohexamer that catalyzes the ATP-dependent formation of phosphorylated acetone and bicarbonate intermediates that react at Mn(II) metal active sites to form acetoacetate. Structural models of X. autotrophicus AC (XaAC) with and without nucleotides reveal that the binding and phosphorylation of the two substrates occurs ~40 Å from the Mn(II) active sites where acetoacetate is formed. Based on the crystal structures, a significant conformational change was proposed to open and close a tunnel that facilitates the passage of reaction intermediates between the sites for nucleotide binding and phosphorylation of substrates and Mn(II) sites of acetoacetate formation. We have employed electron paramagnetic resonance (EPR), kinetic assays, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) of poised ligand-bound states and site-specific amino acid variants to complete an in-depth analysis of Mn(II) coordination and allosteric communication throughout the catalytic cycle. In contrast with the established paradigms for carboxylation, our analyses of XaAC suggested a carboxylate shift that couples both local and long-range structural transitions. Shifts in the coordination mode of a single carboxylic acid residue (αE89) mediate both catalysis proximal to a Mn(II) center and communication with an ATP active site in a separate subunit of a 180 kDa α2β2γ2 complex at a distance of 40 Å. This work demonstrates the power of combining structural models from X-ray crystallography with solution-phase spectroscopy and biophysical techniques to elucidate functional aspects of a multi-subunit enzyme.

Biochemistry & Molecular Biology↗

NASA NTRS2004

Grounding Headphones for Protection Against ESD

A simple alternative technique has been devised protecting delicate equipment against electrostatic discharge (ESD) in settings in which workers wear communication headsets. In the original setting in which the technique was devised, the workers who wear the headsets also wear anti-ESD grounding straps on their wrists. The alternative technique eliminates the need for the wrist grounding straps by providing for grounding through the headsets. In place of the electrically insulating foam pads on the headsets, one installs pads made of electrically conductive foam like that commonly used to protect electronic components. Grounding wires are attached to the conductive foam pads, then possibly to the shielding cable which may be grounded to the backshell on the connector. The efficacy of this technique in protecting against ESD has been verified in experiments. The electrical resistance of the pads is a few megohms - about the same as that of a human body between the fingers of opposite hands and, hence, low enough for grounding. The only drawback of the technique is that care must be taken to place the foam pads in contact with the user s skin: any hair that comes between the foam pads and the skin must be pushed aside because hair is electrically insulating and thus prevents adequate grounding.

Peters, John↗