In situ Spectroscopy on Intact Leptospirillum ferrooxidans Reveals that Reduced Cytochrome 579 is an Obligatory Intermediate in the Aerobic Iron Respiratory Chain.

Robert C Blake

doi:10.3389/fmicb.2012.00136

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.

Highlights

Certain chemolithotrophic bacteria inhabit ore-bearing geological formations exposed to the atmosphere and obtain all of their energy for growth from the oxidation and dissolution of minerals within the ore
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
This paper introduces a new means to study respiratory electron transfer reactions in situ in intact bacteria under physiological conditions

Summary

Introduction

Certain chemolithotrophic bacteria inhabit ore-bearing geological formations exposed to the atmosphere and obtain all of their energy for growth from the oxidation and dissolution of minerals within the ore. Given the genetic diversity within this collection of phenotypically related bacteria, it would not be surprising to learn that phylogenetically distinct groups of bacteria express different electron transfer biomolecules and pathways to accomplish aerobic respiration on soluble iron. Classic reductionist studies that involve the structural and functional characterization of highly purified proteins in dilute solution have described a bewildering variety of different redoxactive electron transport proteins in cell-free extracts derived from iron-grown Gram-negative (Cox and Boxer, 1978; Hart et al, 1991; Blake et al, 1992; Yarzábal et al, 2002, 2004), Gram-positive (Blake et al, 1993; Takai et al, 2001; Dinarieva et al, 2010), and Archaea (Hettmann et al, 1998; Dopson et al, 2005; Auernik and Kelly, 2008) bacteria. The proteins in the aerobic iron respiratory pathway of At. ferrooxidans do not appear to be expressed in many of the phylogenetically distinct bacteria that respire on iron. Comparative analyses conducted using those relevant bacterial genomes where partial or complete DNA sequence data is available www.frontiersin.org

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