Abstract
Cytochrome bc1 is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Qo site in cytochrome b with cytochrome c1. The Qo site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Qo site. Here, using a bacterial system, we show that mutation G167P in cytochrome b shifts the equilibrium distribution of ISP-HD toward positions remote from the Qo site. This renders cytochrome bc1 non-functional in vivo. This effect is remediated by addition of alanine insertions (1Ala and 2Ala) in the neck region of the ISP subunit. These insertions, which on their own shift the equilibrium distribution of ISP-HD in the opposite direction (i.e. toward the Qo site), also act in this manner in the presence of G167P. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc1 to generate superoxide, which becomes evident when the concentration of quinone increases. This result corroborates the recently proposed model in which "semireverse" electron transfer back to the Qo site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. G167P suggests possible molecular effects of S151P (corresponding in sequence to G167P) identified as a mitochondrial disease-related mutation in human cytochrome b. These effects may be valid for other human mutations that change the equilibrium distribution of ISP-HD in a manner similar to G167P.
Highlights
Mutation S151P was found in patients with exercise intolerance
Spectral and Kinetic Properties of G167P in Cytochrome b— As shown in Fig. 1C, Gly-167 in R. capsulatus cytochrome b is located at the end of helix cd1 close to the twist loop that separates this helix from helix cd2
G167P mutant is not functional in vivo as indicated by the incapability of cells to sustain cytochrome bc1-dependent photosynthetic growth (Fig. 3 and Table 1). This correlates with low enzymatic activity of the mutant and severe impediments in the operation of the Qo site identified by light-induced kinetic measurements
Summary
Mutation S151P was found in patients with exercise intolerance. Results: Bacterial analogous substitution (G167P) influences movement of the iron-sulfur protein head domain (ISP-HD), increasing ROS production. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc to generate superoxide, which becomes evident when the concentration of quinone increases This result corroborates the recently proposed model in which “semireverse” electron transfer back to the Qo site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. Under certain conditions, part of the energy released from oxidation of quinol can be dissipated through side reactions that result in partial or total loss of proton translocation function of cytochrome bc1 These reactions reduce the efficiency of separation of electrons in the Qo site into two cofactor chains. This allowed us to extend the array of conditions that tested changes in the levels of superoxide production by cytochrome bc toward a better understanding of the mechanism of this reaction
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