In situ spectroscopy on intact Leptospirillum ferrooxidans reveals that reduced cytochrome 579 is an obligatory intermediate in the aerobic iron respiratory chain

Abstract

Electron transfer reactions among colored cytochromes in intact bacterial cells were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scatter light. The aerobic iron respiratory chain of Leptospirillum ferrooxidans was dominated by the redox status of an abundant cellular cytochrome that had an absorbance peak at 579 nm in the reduced state. Intracellular cytochrome 579 was reduced within the time that it took to mix a suspension of the bacteria with soluble ferrous iron at pH 1.7. Steady state turnover experiments were conducted where the initial concentrations of ferrous iron were less than or equal to that of the oxygen concentration. Under these conditions, the initial absorbance spectrum of the bacterium observed under air-oxidized conditions was always regenerated from that of the bacterium observed in the presence of Fe(II). The kinetics of aerobic respiration on soluble iron by intact L. ferrooxidans conformed to the Michaelis–Menten formalism, where the reduced intracellular cytochrome 579 represented the Michaelis complex whose subsequent oxidation appeared to be the rate-limiting step in the overall aerobic respiratory process. The velocity of formation of ferric iron at any time point was directly proportional to the concentration of the reduced cytochrome 579. Further, the integral over time of the concentration of the reduced cytochrome was directly proportional to the total concentration of ferrous iron in each reaction mixture. These kinetic data obtained using whole cells were consistent with the hypothesis that reduced cytochrome 579 is an obligatory steady state intermediate in the iron respiratory chain of this bacterium. The capability of conducting visible spectroscopy in suspensions of intact cells comprises a powerful post-reductionist means to study cellular respiration in situ under physiological conditions for the organism.

New Group in the Leptospirillum Clade: Cultivation-Independent Community Genomics, Proteomics, and Transcriptomics of the New Species “Leptospirillum Group IV UBA BS”

“…Cytochrome 579 (Cyt 579 ) is involved in electron transport (54,55). The presence of a multicopper oxidase, as well as Cyt 572 and Cyt 579 , whose biochemical function was verified in Leptospirillum group II from the Richmond Mine (52,54), provides strong evidence supporting the role of Leptospirillum group IV UBA BS in iron oxidation.…”

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

“…Leptospirillum group III proteins involved in the various steps of the iron oxidation electron transport chain (Jeans et al, 2008;Singer et al, 2008;Blake and Griff, 2012;Bonnefoy and Holmes, 2012) differed in their response to temperature. Three cytochromes involved in the initial steps of iron oxidation were more abundant at 46 1C than at 40 1C (two of which were significantly upregulated: Cyt 579 P ¼ 0.05; cytochrome C P ¼ 0.03).…”

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant

“…Under those conditions, the absorbed radiant power should be independent of scattering effects (27)(28)(29)(30). Equilibrium and kinetic studies were conducted on the Fe(II)-dependent reduction and O 2 -dependent oxidation of cytochromes in intact Leptospirillum ferrooxidans at pH 1.7 (26). A cytochrome with a reduced spectral peak at 579 nm was shown to be an obligatory intermediate in the aerobic iron respiratory chain of L. ferrooxidans.…”

“…This laboratory exploited an integrating cavity absorption meter to study respiratory electron transfer reactions in situ in intact bacteria under physiological conditions (26). The observation chamber of this novel spectrophotometer included a reflecting cavity completely filled with the absorbing suspension.…”