Abstract

Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm(2) in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s(-1). The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment.

Highlights

  • The order of electron transfer in the respiratory chain of Acidithiobacillus ferrooxidans is unknown

  • The observations described above were consistent with the hypothesis that rusticyanin and cytochromes a and c were the principal participants in the aerobic iron respiratory chain of At. ferrooxidans

  • Our initial hope was that we could observe the sequential reduction of these electron transport proteins in their hypothesized linear array

Read more

Summary

Background

The order of electron transfer in the respiratory chain of Acidithiobacillus ferrooxidans is unknown. The colored prosthetic groups of most electron transport proteins include intrinsic spectrophotometric probes whereby transient changes in the oxidation-reduction state of the proteins may be monitored with great sensitivity This laboratory exploited an integrating cavity absorption meter to study respiratory electron transfer reactions in situ in intact bacteria under physiological conditions [26]. The premise was that accurate UV-visible spectroscopy of electron transfer reactions among colored proteins could be conducted in highly turbid suspensions if the live bacteria were irradiated in an isotropic homogeneous field of incident measuring light Under those conditions, the absorbed radiant power should be independent of scattering effects [27,28,29,30]. An unexpected outcome was the demonstration that, despite their different electrochemical potentials in vitro, the three principal types of redox-active proteins in the iron respiratory chain behaved as a single ensemble in situ

Experimental Procedures
Results
Discussion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call