Abstract
The exact role that cytochrome 579 plays in the aerobic iron respiratory chain of Leptospirillum ferriphilum is unclear. This paper presents genomic, structural, and kinetic data on the cytochrome 579 purified from cell-free extracts of L. ferriphilum cultured on soluble iron. Electrospray mass spectrometry of electrophoretically homogeneous cytochrome 579 yielded two principal peaks at 16,015 and 16,141 Daltons. N-terminal amino acid sequencing of the purified protein yielded data that were used to determine the following: there are seven homologs of cytochrome 579; each homolog possesses the CXXCH heme-binding motif found in c-type cytochromes; each of the seven sequenced strains of L. ferriphilum expresses only two of the seven homologs of the cytochrome; and each homolog contains an N-terminal signal peptide that directs the mature protein to an extra-cytoplasmic location. Static light scattering and macroion mobility measurements on native cytochrome 579 yielded masses of 125 and 135 kDaltons, respectively. The reduced alkaline pyridine hemochromogen spectrum of the purified cytochrome had an alpha absorbance maximum at 567 nm, a property not exhibited by any known heme group. The iron-dependent reduction and oxidation of the octameric cytochrome exhibited positively cooperative kinetic behavior with apparent Hill coefficients of 5.0 and 3.7, respectively, when the purified protein was mixed with mM concentrations of soluble iron. Consequently, the extrapolated rates of reduction at sub-mM iron concentrations were far too slow for cytochrome 579 to be the initial iron oxidase in the aerobic respiratory chain of L. ferriphilum. Rather, these observations support the hypothesis that the acid-stable cytochrome 579 is a periplasmic conduit of electrons from initial iron oxidation in the outer membrane of this Gram-negative bacterium to a terminal oxidase in the plasma membrane.
Highlights
Leptospirillum ferrooxidans (Markosyan, 1972; Hippe, 2000), Leptospirillum ferriphilum (Coram and Rawlings, 2002), and Leptospirillum ferrodiazotrophum (Tyson et al, 2005) are species of mesophilic, vibrioid, or spiral-shaped Gram-negative prokaryotes that can be acquired as pure cultures from national/ commercial culture collections
Prior reductionist studies reported that cell-free extracts derived from iron-grown L. ferrooxidans (Hart et al, 1991), L. ferriphilum (Ram et al, 2005), or a microbial biofilm community with a low diversity of microbes that was dominated by Leptospirillum group II bacteria (Singer et al, 2008) all contained readily discernible quantities of an acid-stable, acid-soluble cytochrome with an unusual absorbance maximum at 579 nm in the reduced state
This paper describes selected structural and functional properties of cytochrome 579 that was purified to electrophoretic homogeneity from cell-free extracts of the type strain of L. ferriphilum
Summary
Leptospirillum ferrooxidans (Markosyan, 1972; Hippe, 2000), Leptospirillum ferriphilum (Coram and Rawlings, 2002), and Leptospirillum ferrodiazotrophum (Tyson et al, 2005) are species of mesophilic, vibrioid, or spiral-shaped Gram-negative prokaryotes that can be acquired as pure cultures from national/ commercial culture collections. Other potential species in that genus, Leptospirillum rubarum (Goltsman et al, 2009), Leptospirillum thermoferrooxidans (Golovacheva et al, 1992; Hippe, 2000), and a group designated as “Leptospirillum group IV UBA BS” (Goltsman et al, 2013), have been reported, but none of these strains is generally available at the present time All of these organisms are obligately acidophilic chemoautotrophs that are only known to respire aerobically on reduced soluble iron or ferrous iron-containing sulfide minerals to obtain biochemical energy, making them among the most metabolically restricted organisms known. The likely location of this acid-compatible cytochrome 579 was in the acidic periplasm, incomplete reduction of the protein was noted using 30 mM Fe(II) at pH 2.0 Another novel cytochrome with a unique reduced absorbance maximum at 572 nm was purified from the same biofilm communities (Jeans et al, 2008). Cytochrome 579 isolated from early-stage biofilms was much more reactive with Fe(II) at low pH than was cytochrome 579 isolated from
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