Laser Flash-Induced Kinetic Analysis of CytochromefOxidation by Wild-Type and Mutant Plastocyanin from the CyanobacteriumNostocsp. PCC 7119

Abstract

Oxidation of the soluble, truncated form of cytochrome f by wild-type and mutant species of plastocyanin has been analyzed by laser flash absorption spectroscopy in the cyanobacterium Nostoc (formerly, Anabaena) sp. PCC 7119. At low ionic strengths, the apparent electron transfer rate constant of cytochrome f oxidation by wild-type plastocyanin is 1.34 x 10(4) s(-)(1), a value much larger than those determined for the same proteins from other organisms. Upon site-directed mutagenesis of specific residues at the plastocyanin interaction area, the rate constant decreases in all cases yet to varying extents. The only exception is the D54K variant, which exhibits a higher reactivity toward cytochrome f. In most cases, the reaction rate constant decreases monotonically with an increase in ionic strength. The observed changes in the reaction mechanism and rate constants are in agreement with the location of the mutated residues at the interface area, as well as with the peculiar orientation of the two partners within the Nostoc plastocyanin-cytochrome f transient complex, whose NMR structure has been determined recently. Furthermore, the experimental data herein reported match well the kinetic behavior exhibited by the same set of plastocyanin mutants when acting as donors of electrons to photosystem I [Molina-Heredia, F. P., et al. (2001) J. Biol. Chem. 276, 601-605], thus indicating that the copper protein uses the same surface areas-one hydrophobic and the other electrostatic-to interact with both cytochrome f and photosystem I.

A pulsed EPR method to determine distances between paramagnetic centers with strong spectral anisotropy and radicals: The dead-time free RIDME sequence

“…The pEAF-wt [22] expression plasmid containing the sequence encoding the soluble domain of cyt f from Nostoc sp. PCC7119 has been kindly provided by the group of Prof. Miguel De la Rosa, Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla, Spain.…”

“…To improve the maturation and correct insertion of the heme group, Escherichia coli strain MV1190 cells (Bio-Rad) were co-transformed with plasmids pEC86 [22] and the cyt f expression plasmids. The cells were incubated on Luria-Bertani (LB) medium plates (added by 20 mg/L ampicillin, 20 mg/L chloramphenicol) at 37°C for 24 h. Several pre-cultures were incubated in 100 mL flasks with 20 mL of LB medium supplemented with 20 mg/L ampicillin (amp) and 20 mg/L chloramphenicol (cam) at 37°C and 250 rpm for 5-6 h. The pre-cultures with the best OD 600 were used to inoculate 1.7 L (in 5 L Erlenmeyer flasks) in the same medium and incubated at 25°C and 150 rpm for >72 h under semi-anaerobic conditions and high antibiotics pressure by adding further amp and cam after 20 and 40 h. Induction was achieved 20 h after the inoculation of the large culture using 1 mM IPTG.…”

Electrostatic strain and concerted motions in the transient complex between plastocyanin and cytochrome f from the cyanobacterium Phormidium laminosum

“…The electron transfer from Cf to Pc has been analysed by using stopped-flow [11][12][13][14][15] and fast kinetic techniques [7,16], often combined with site-directed mutagenesis of one or both partners. The resulting kinetic data have been very useful to analyse the interaction between Pc and Cf by means of theoretical methods, including Molecular Dynamics (MD) at sub-nanosecond time scale [17], rigid-body MD [18], Molecular Modelling [19] supported by NMR data and Brownian Dynamics (BD) simulations [20][21][22][23][24][25][26][27][28].…”

Cytochrome c 6-like protein as a putative donor of electrons to photosystem I in the cyanobacterium Nostoc sp. PCC 7119

“…PCC 7119, Cyt c 6 and Pc, whose E m at pH 7.0 are ?337 and ?355 mV respectively (Molina-Heredia et al 1998), take electrons from Cyt f, whose E m , at the same pH 7.0, is ?334 mV (Albarrán et al 2005), and donate them to the photo-oxidized PSI, whose E m (P700) is ?500 mV (Brettell 1997). The E m at pH 7.0 of the Nostoc Cyt c 6 -like protein is ?199 mV ( (Bialek et al 2008), that is one other Cyt c 6 -like protein.…”

“…electron transfer chain, namely petJ, petE(Molina-Heredia et al 1998), and petA(Albarrán et al 2005).…”