Electron transfer and spectral alpha-band properties of the di-heme protein cytochrome c4 from Pseudomonas stutzeri.

Cytochrome c4 is a 190-residue protein active in the aerobic and anaerobic respiration of several bacteria. We have isolated Pseudomonas stutzeri (ATCC no. 11607) cytochrome c4 by an optimized growth procedure following factorial design. The ultraviolet/visible spectra of reduced cytochrome c4 have a composite alpha/beta band which can be resolved into six components. One of these seems to be specific for the high-potential heme group. The kinetics for full oxidation and reduction with the two inorganic redox couples, [Co(terpy)2]2+/3+ and [Co(bipy)3]2+/3+, is formally compatible with either bi- or tri-exponential kinetics. The former would be in line with weak interaction between the heme groups, the latter with notable interaction effects. Arguments in favour of the latter and a cooperative two-electron transfer pattern are given. All phases are approximately proportional to the Co-complex concentration, implying that intramolecular electron transfer in this time range is unlikely. The rate constants are in the range (0.7-80) x 10(4) M-1 s-1 at pH = 7.6 (Tris) and 0.1 M NaCl and very little dependent on the ionic strength in the range 0.1-0.3 M. The reduction potentials could be calculated from the forward and reverse rate constant ratios. The values are 241 +/- 5 and 328 +/- 2 mV (Nernst hydrogen electrode) if bi-exponential kinetics is used and interaction between the heme groups disregarded. The intrinsic microscopic reduction potential values are closer when the tri-exponential, cooperative model is used as this model transfers 30-40 mV to electrostatically dominated interaction potentials. The overall electron transfer pattern can be related to the recently determined crystal structure of the P. stutzeri cytochrome c4.

Redox potential and electrostatic effects in competitive inhibition of dual-path electron transfer reactions of spinach plastocyanin.

Redox inactive ions with high positive charges lower the rate constant for oxidation of several plant plastocyanins (PC) by small positively charged inorganic reaction partners. The rate constant decrease is commonly attributed to competitive inhibition where the redox inactive ions are bound to the negatively charged remote electron transfer (ET) site of PC and block this site sterically. We have investigated the effects of the inhibitor [NH3)5Co(NH2)Co(NH3)5]5+ on the ET reactions of spinach PC with [Co(phen)3]3+ (phen = 1,10-phenanthroline) and the electrically neutral analogue [Co(phen-SO3)3] (phen-SO3 = 5-sulfonato-1,10-phenanthroline) at the ionic strengths mu = 0.1 M and 0.03 M. Inhibition of the [Co(phen)3]3+ reactions is notably smaller for PC(II) reduction than for PC(I) oxidation. This is indicative of a redox potential increase of PC(II)/PC(I) on inhibitor attachment. The effect amounts to 16 mV at mu = 0.1 M and 31 mV at mu = 0.03 M. These data, and analysis in terms of ET theory show that inhibition cannot be caused solely by steric blocking. Driving force and interreactant electrostatic work terms are equally important. The PC(I)/[Co(phen-SO3)3] reaction exhibits a more entangled pattern. The rate constant first increases slightly with increasing inhibitor concentration, then drops, and approaches a constant value not far from the original value. This pattern is in line with association between the negatively charged -SO3- groups of the Co(III) complex and the inhibitor, and ET of the associate at both ET sites of PC.

Resonance effects in strongly exothermic long-range electron transfer and their possible implications for the behaviour of site-directed mutant proteins.

Long-range electron transfer investigations of hemoproteins, blue copper and iron-sulphur proteins frequently rest on electronically excited metal centres. When the excitation energy approaches the oxidation or reduction potentials of intermediate residues the superexchange view normally used, however, fails and a variety of new dynamic features arise. These all involve population of the intermediate cation or anion residue states which can be partially or wholly vibrationally relaxed. We discuss suitable views and a new theoretical formalism for these phenomena. We also note some important implications for site-directed mutagenesis in long-range, strongly exothermic electron transfer processes.

Effects of NO2-modification of Tyr83 on the reactivity of spinach plastocyanin with cytochrome f.

We have investigated the electron transfer (ET) reactions between turnip cytochrome f, and the native and NO2-Tyr83-modified forms of spinach plastocyanin (PCu) at 10.0 degrees C and ionic strength 0.200 M(NaCl), in both directions as a function of pH. The PCu(II)/cytochrome f(II) rate constants in the pH-range 4-6.8 reflect active and remote binding site protonation. At higher pH, NO2-Tyr83 and positively charged residues on cytochrome f are deprotonated, and both native and NO2-modified PCu exhibit a composite rate constant variation in this pH range. When framed by ET theory this pattern is fully understandable in terms of variations in reduction potentials and electrostatic interactions, caused by the protonation equilibria. The rate constant ratio knitro/knative is, however, only 1.04 for the PCu(II)/cytochrome f(II) reactions in spite of a 18 mV higher reduction potential for NO2-Tyr83-modified PCu. This is much lower than the value of 1.42 expected from ET theory solely on the basis of such a reduction potential effect. A similar effect is seen for PCu(I)/cytochrome f(III) for which the low-pH knitro/knative ratio is 0.51. Notable but smaller effects are also observed for the small reaction partners [Fe(CN)6]3-/4- and [Co(phen)3]3+/2+. The effect of NO2-modification in addition to the reduction potential effect can be resolved into a small reorganization energy increase around the copper atom and a smaller electronic transmission coefficient for ET through the Cu/Cys84/Tyr83 sequence. The former effect dominates in the reactions with the small reaction partners, while the electronic effects contribute significantly for PCu/cytochrome f, supporting the concept that the PCu/cytochrome f ET is at the remote PCu binding site.

Isolation and purification of plastocyanin from spinach stored frozen using hydrophobic interaction and ion-exchange chromatography.

A new simple three-day procedure for preparative isolation and purification of plastocyanin from spinach stored in the frozen state is described. This procedure is based on batch adsorption on ion-exchange resin, ammonium sulphate precipitation, and purification on a Phenyl-Sepharose hydrophobic interaction column and a single Q Sepharose High Performance ion-exchange column. Approximately 100 mg of plastocyanin with an absorbance ratio A278/A597 of 1.10±0.02 in the oxidized state was typically obtained from 12 kg of spinach leaves. The purified spinach plastocyanin is shown to be homogeneous to the resolution of free solution capillary electrophoresis.

A new procedure for fast isolation and purification of plastocyanin from the cyanobacterium Anabaena variabilis.

Methods are described for growing the cyanobacterium A. variabilis and for the isolation and purification of plastocyanin from the grown culture. Cell paste which had been stored at -35°C was suspended in 1 mM MES buffer, pH 6.5 and centrifuged. The supernatant was diluted to a conductivity of 0.12 mS, [Fe(CN)6](3-) added to a concentration of 0.5 mM and the solution loaded on a S Sepharose Fast Flow column. After elution and ultrafiltration, the plastocyanin containing fractions were reloaded on a S Sepharose Fast Flow column for final purification. A typical yield in three days from cells harvested from 3×20 l of medium was 32 mg plastocyanin with a minimum absorbance ratio A278/A597=1.14. This procedure is faster and the yield higher than for previous procedures.