Proton Transfer in the K-Channel Analog of B-Type Cytochrome c Oxidase from Thermus thermophilus

Abstract

A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers.

Dynamics of the KB Proton Pathway in Cytochrome ba3 from Thermus thermophilus

“…[38] Glu-286 is located at the 'top' of the D-pathway in the A-type oxidases,~10 Å from Cu B , whereas the Glu-15 II in the ba 3 oxidase is located~26 Å from Cu B . The importance of the Glu-15 II residue in the ba 3 oxidase is consistent with a molecular dynamics (MD) study of the K B pathway, [43] which showed that Glu-15 II can adopt two conformations; in the protonated state it points inward into the continuation of the pathway, connecting to Thr-315 (see Figure 1), whereas in the deprotonated state it can adopt a conformation where it instead points towards bulk water. [20]).…”

“…This transition in the WT oxidase shows no pH dependence in the corresponding pH range ( Figure 6 and [26]). Such a water molecule very close to Y248 (HOH-102 or 630) is seen in the crystal structure (see [14] and Figure 1B) and remains stable in the position bridging between Y248 and T315 in simulations (W630 in [43]). In such a scenario, the results with the Y248T variant can be explained by a shift in the pK a of this internal group from > 10 to~8.8.…”

“…Formation of A is followed by electron transfer from heme b (heme a in R. sphaeroides aa 3 ) to the catalytic site with a time constant of 15 ms or~40 ms in the ba 3 and aa 3 , respectively. Also shown is a resolved H 2 O molecule (HOH-630 (or 102)) as well as residues Thr-302 and Asp-372 suggested to be involved in the path for pumped protons (Thr-302 [43] ) and in defining the PLS (Asp-372 [22,23] ). Also shown is a resolved H 2 O molecule (HOH-630 (or 102)) as well as residues Thr-302 and Asp-372 suggested to be involved in the path for pumped protons (Thr-302 [43] ) and in defining the PLS (Asp-372 [22,23] ).…”

Water exit pathways and proton pumping mechanism in B-type cytochrome c oxidase from molecular dynamics simulations

“…[28] Although not sharing any significant sequence homology, this channel is located at a similar position as the K-channel in A-type CcO and is therefore referred to as the K-channel analog. While this proton uptake channel has been investigated in detail, [29] the means by which the protons exit the enzyme remain unclear. [30]…”

pKA in proteins solving the Poisson–Boltzmann equation with finite elements

“…Titratable groups of a protein can be located at the surface or be buried. Their protonation state plays an important role in proton translocation through membrane proteins as for instance proton pumps or proton channels . With the three‐dimensional crystal structure of a protein, it is possible to compute the electrostatic energy parameters necessary to determine the protonation of titratable residues by solving the linear Poisson–Boltzmann equation ( l PBE) .…”