A single-amino-acid lid renders a gas-tight compartment within a membrane-bound transporter

Abstract

Proteins undergo structural fluctuations between nearly isoenergetic substates. Such fluctuations are often intimately linked with the functional properties of proteins. However, in some cases, such as in transmembrane ion transporters, the control of the ion transport requires that the protein is designed to restrict the motions in specific regions. In this study, we have investigated the dynamics of a membrane-bound respiratory oxidase, which acts both as an enzyme catalyzing reduction of O 2 to H2O and as a transmembrane proton pump. The segment of the protein where proton translocation is controlled (''gating'' region) overlaps with a channel through which O 2 is delivered to the catalytic site. We show that the replacement of an amino acid residue with a small side chain (Gly) by one with a larger side chain (Val), in a narrow part of this channel, completely blocks the O 2 access to the catalytic site and results in formation of a compartment around the site that is impermeable to small gas molecules. Thus, the protein motions cannot counter the blockage introduced by the mutation. These results indicate that the protein motions are restricted in the proton-gating region and that rapid O 2 delivery to the catalytic site requires a gas channel, which is confined within a rigid protein body.membrane protein ͉ cytochrome c oxidase ͉ proton transfer ͉ dynamics ͉ respiratory oxidases

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

“…Thus, also in bacteriorhodopsin, a large KIE is associated only with proton transfer across the pumping element. As pointed out by Wikström (47) a large KIE may be characteristic for proton transfer through a well ordered structure of protonatable groups such as that expected to surround the pumping element (48).…”

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…As far as redox enzymes are concerned, the best documented example is acetyl-CoA synthase/CO dehydrogenase, [434][435][436][437] but channels are also suspected in NiFe hydrogenases (Figure 1), [438][439][440] FeFe hydrogenases, 439,441 cytochrome c oxidase, 442,443 copper-containing amine oxidase, 444 and Photosystem II. 445 However, one may also consider that small diatomic molecules are able to freely diffuse through the protein matrix and, therefore, the requirement for a channel to transport CO, H 2 , O 2 , and other small substrates is questionable.…”

Experimental approaches to kinetics of gas diffusion in hydrogenase

“…For example, certain mutations in enzymes that use molecular oxygen as substrate increase the K m for O 2 (4,5,8,15). Brzezinski and coworkers (6) demonstrated that a glycine-to-valine mutation almost completely obstructs the oxygen channel of cytochrome c oxidase; in this study, the delayed access of substrate O 2 and inhibitor CO to the active-site heme was probed by using time-resolved UV-vis spectroscopy. From these observations, they inferred that the protein is rigid in the region of this residue, because otherwise fluctuations would counter the blockage introduced by the mutation.…”

“…As far as redox catalysis is concerned, the best-documented example is certainly the 70-Å-long channel that connects the two active sites involved in CO production and utilization in acetyl-CoA synthase/CO dehydrogenase (ACS-CODH) (2,3). In addition, channels dedicated to the transport of O 2 , N 2 , or H 2 are supposed to exist in hemecopper oxidase (4)(5)(6), nitrogenase (7), lipoxygenase (8), photosystem II (9), and both NiFe and FeFe hydrogenases (10), to cite but a few. These channels were often discovered by searching for hydrophobic cavities in x-ray structures hence their qualification as ''static.''…”