Modifications of Protein Environment of the [2Fe-2S] Cluster of the bc1 Complex

Abstract

The rate-determining step in the overall turnover of the bc 1 complex is electron transfer from ubiquinol to the Rieske ironsulfur protein (ISP) at the Q o -site. Structures of the ISP from Rhodobacter sphaeroides show that serine 154 and tyrosine 156 form H-bonds to S-1 of the [2Fe-2S] cluster and to the sulfur atom of the cysteine liganding Fe-1 of the cluster, respectively. These are responsible in part for the high potential (E m,7 ϳ300 mV) and low pK a (7.6) of the ISP, which determine the overall reaction rate of the bc 1 complex. We have made site-directed mutations at these residues, measured thermodynamic properties using protein film voltammetry to evaluate the E m and pK a values of ISPs, explored the local proton environment through two-dimensional electron spin echo envelope modulation, and characterized function in strains S154T, S154C, S154A, Y156F, and Y156W. Alterations in reaction rate were investigated under conditions in which concentration of one substrate (ubiquinol or ISP ox ) was saturating and the other was varied, allowing calculation of kinetic terms and relative affinities. These studies confirm that H-bonds to the cluster or its ligands are important determinants of the electrochemical characteristics of the ISP, likely through electron affinity of the interacting atom and the geometry of the H-bonding neighborhood. The calculated parameters were used in a detailed Marcus-Brønsted analysis of the dependence of rate on driving force and pH. The proton-first-then-electron model proposed accounts naturally for the effects of mutation on the overall reaction.Enzymes of the cytochrome bc 1 complex 2 family (EC 1.10.2.2) are ubiquitous membrane proteins in mitochondria and bacteria (and, including the b 6 f complex, in chloroplasts) that play a central role in the electron transfer chains of respiration and photosynthesis. In Rhodobacter sphaeroides, the bc 1 complex, together with the reaction center (RC) and cytochrome (cyt) c 2 , forms the photosynthetic chain. The complex catalyzes oxidation of substrate ubiquinol (QH 2 ) by a watersoluble ferricytochrome c 2 in the periplasmic space and generates a proton-motive force for ATP synthesis through a Q-cycle mechanism (1, 2). With the availability of structures from mitochondrial and bacterial systems (3-8), the mechanism could be further refined. Proteins of the bc 1 complex family form homodimers inserted in the cellular or mitochondrial membrane, each monomer containing minimally the three subunits (cyt b with two b-type hemes (b H and b L ), cyt c 1 with heme c 1 , and Rieske-type iron-sulfur protein (ISP)) that form the catalytic core of the complex. The ␣-proteobacterial ancestors of mitochondria were likely derived from photosynthetic progenitors, accounting for the high degree of homology in sequence, structure, and mechanism. The bacterial structures are much simpler than the 8 -11 subunit complexes in mitochondria. The isolated complex has four subunits in each monomer (cyt b, cyt c 1 , ISP, and subunit IV) (9, 10), but crystallo...

Quinol oxidase encoded by cyoABCD in Rhizobium etli CFN42 is regulated by ActSR and is crucial for growth at low pH or low iron conditions

“…The premise is based on several studies in another alphaproteobacterium, Rhodobacter sphaeroides, where Fbc activity was optimum at pH 8.0 and dropped to a relatively low level at pH v6.0 (Crofts et al, 1999;Guergova-Kuras et al, 2000;Lhee et al, 2010;Zhou et al, 2012). If these in vitro conditions are relevant to changes the pH of the growth medium, an acidic pH would cause lower Fbc activity leading to a higher quinol : quinone ratio and, consequently, greater ActSR activity and higher levels of cyo expression.…”

Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc1 complex

“…(1)). The solid lines in show F i (X ) based on λ = 3.99 eV from Anton simulations, λ S t = 1.46 eV from separate MD runs of 200 ns long, and ∆F 0 = 0.15 eV from experiment 9. The dashed lines are the result of the standard model 2 assuming λ = λ St = 1.46 eV.…”

“…1(b)), was suggested as the initial state from which electron is transferred to Fe 2 S 2 . 7, 9 We have previously studied this configuration by molecular dynamics (MD) simulations but found the length of trajectories (∼200 ns) insufficient to converge the parameters needed to calculate the rate. 10 Here, we present new 13 µs simulation data produced on the Anton supercomputer.…”

“…An additional insight can be gained from the energy gap law, 2 i.e., to the dependence of the rate on the reaction free energy ∆F 0 experimentally achieved by mutagenesis. 9 These data are shown in Fig. 5 where the rates of electron transfer k ET in different mutants relative to the rate in the wild type (WT) protein k WT are plotted against experimental ∆F 0 .…”

“…λ is provided by our Anton simulations (Table I) and ∆F 0 = 0.15 eV is taken from experiment. 9 In addition, since ⟨X⟩ i converge much faster than λ, we have calculated λ St = 1.46 eV (κ G = 2.7) from two separate 200 ns trajectories. More detail on the simulation protocol is provided in the supplementary material, 15 and we now turn to the mechanistic question of what are the nuclear modes and interactions affecting X.…”