Charge separation in Rhodobacter sphaeroides mutant reaction centers with increased midpoint potential of the primary electron donor

Abstract

Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll B(A) at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P(+)H(A)(-) state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P(+)B(A)(-)H(A) and P(+)B(A)H(A)(-) states. The data give grounds for assuming that the value of the energy difference between the states P*, P(+)B(A)(-)H(A), and P(+)B(A)H(A)(-) at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll B(A) is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P(+)B(A)(-) state with respect to P*.

High efficiency excitation energy transfer in biohybrid quantum dot–bacterial reaction center nanoconjugates

“…This long-lived bleach is likely a signature of a very small percentage of RCs that were able to undergo charge-separation. This combination of spectral features was consistent with the expected effects of combining the triple H-bond [36][37][38] and β-mutations, 39,40 very strongly reducing the quantum yield of charge separation and greatly increasing the lifetime of the P* excited state from ~3 ps to over 150 ps.…”

“…35 To use TCSPC to determine the efficiency of EET in RC-QD nanoconjugates, a new RC mutant was engineered, 3Hβ (Figure 1(a)), that retains a normal long-wavelength primary electron donor but has a drastically-reduced quantum yield for charge separation (see SI, Section 1). This was achieved by combining a triple mutation LL131H + LM160H + FM197H, [36][37][38] with the so-called 'b-mutation' LM214H. 39,40 The triple mutation adds three hydrogen bonds between the protein scaffold and the two bacteriochlorophylls that make up P, raising its mid-point potential for one electron oxidation by around 260 mV but without markedly affecting its absorption spectrum.…”

“…37 This has the effect of raising the free energy associated with the first radical pair formed during charge separation, P + BA -, above that of the excited singlet state of P (P*), thus severely slowing primary charge separation (causing a ~50% decrease in quantum yield) and enhancing the fluorescence of P* through an extension of its lifetime from ~3 ps to ~50 ps. 38 The 'b-mutation' replaces the bacteriopheophytin that is the second acceptor during charge separation (HA) with a bacteriochlorophyll (bA) (Figure 1a). 39,40 In isolation this mutation renders the first (BA) and second (bA) electron acceptor isoenergetic, enabling formation of an admixture of the P + BAand P + bAradical pairs.…”

X-ray structure of the Rhodobacter sphaeroides reaction center with an M197 Phe→His substitution clarifies the properties of the mutant complex

“…The F(M197)H mutation was introduced using the genetic system for site-directed mutagenesis, as described previously (Khmelnitskiy et al, 2013). An altered pufM gene was shuttled into the broad-host-range vector, a derivative of pRK415, containing an EcoRI-HindIII DNA fragment that included the pufBALMX genes.…”

“…It is known that additional hydrogenbonding networks can provide new electrontransfer pathways in proteins (Beratan et al, 1991). Changes in the electron-conducting properties of a protein in the proximity of the primary electron donor and the nearest electron acceptor might be a possible reason for the unpredictably high rates of primary charge separation and quantum yields of this process reported for F(M197)R and F(M197)H mutant RCs, in contrast to the other known hydrogen-bond mutants, which demonstrated decreased rates of forward electron transfer (Ridge et al, 2000;Wang et al, 2007;Khmelnitskiy et al, 2013).…”