skip to main content


Title: Homogeneous Cytochrome 579 Is an Octamer That Reacts Too Slowly With Soluble Iron to Be the Initial Iron Oxidase in the Respiratory Chain of Leptospirillum ferriphilum
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.  more » « less
Award ID(s):
1830866
NSF-PAR ID:
10283511
Author(s) / Creator(s):
; ; ;
Date Published:
Journal Name:
Frontiers in Microbiology
Volume:
12
ISSN:
1664-302X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Atomi, Haruyuki (Ed.)
    Proteins that oxidize extracellular substrates in Gram-positive bacteria are poorly understood. Ferrimicrobium acidiphilum is an actinobacterium that respires aerobically on extracellular ferrous ions at pH 1.5. In situ absorbance measurements were conducted on turbid suspensions of intact Fm. acidiphilum using an integrating cavity absorption meter designed for that purpose. Initial velocity kinetic studies monitored the appearance of product ferric ions in the presence of catalytic quantities of cells. Cell-catalyzed iron oxidation obeyed the Michaelis-Menten equation with values for KM and Vmax of 71 µM and 0.29 fmol/min/cell, respectively. Limited-turnover kinetic studies were conducted with higher concentrations of cells to detect and monitor changes in the absorbance properties of cellular redox proteins when the cells were exposed to limited quantities of soluble reduced iron. A single a-type cytochrome with reduced absorbance peaks at 448 and 605 nm was the only redox-active chromophore that was visible as the cells respired aerobically on iron. The reduced cytochrome 605 exhibited mathematical and correlational properties that were consistent with the hypothesis that oxidation of the cytochrome constituted the rate-limiting step in the aerobic respiratory process with a turnover number of 35 ± 2 s-1. Genomic and proteomic analyses showed that Fm. acidiphilum could and did express only two a-type heme copper terminal oxidases. Cytochrome 605 was associated with the terminal oxidase gene that is located between nucleotides 31090 and 33039, inclusive, in the annotated circular genome of this bacterium. 
    more » « less
  2. Komeili, Arash (Ed.)
    Iron (Fe) oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2 PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism. IMPORTANCE Iron is practically ubiquitous across Earth’s environments, central to both life and geochemical processes, which depend heavily on the redox state of iron. Although iron oxidation, or “rusting,” can occur abiotically at near-neutral pH, we find neutrophilic iron-oxidizing bacteria (FeOB) are widespread, including in aquifers, sediments, hydrothermal vents, pipes, and water treatment systems. FeOB produce highly reactive Fe(III) oxyhydroxides that bind a variety of nutrients and toxins; thus, these microbes are likely a controlling force in iron and other biogeochemical cycles. There has been mounting evidence that Cyc2 functions as an iron oxidase in neutrophiles, but definitive proof of its function has long eluded us. This work provides conclusive biochemical evidence of iron oxidation by Cyc2 from neutrophiles. Cyc2 is common to a wide variety of iron oxidizers, including acidophilic and phototrophic iron oxidizers, suggesting that this fused cytochrome-porin structure is especially well adapted for iron oxidation. 
    more » « less
  3. Newton, Ryan J. (Ed.)
    ABSTRACT

    The iron-oxidizing Gallionellaceae drive a wide variety of biogeochemical cycles through their metabolisms and biominerals. To better understand the environmental impacts of Gallionellaceae, we need to improve our knowledge of their diversity and metabolisms, especially any novel iron oxidation mechanisms. Here, we used a pangenomic analysis of 103 genomes to resolve Gallionellaceae phylogeny and explore their genomic potential. Using a concatenated ribosomal protein tree and key gene patterns, we determined Gallionellaceae has four genera, divided into two groups: iron-oxidizing bacteria (FeOB)Gallionella,Sideroxydans, andFerriphaseluswith iron oxidation genes (cyc2,mtoA) and nitrite-oxidizing bacteria (NOB)CandidatusNitrotoga with the nitrite oxidase genenxr. The FeOB and NOB have similar electron transport chains, including genes for reverse electron transport and carbon fixation. Auxiliary energy metabolisms, including S oxidation, denitrification, and organotrophy, were scattered throughout the FeOB. Within FeOB, we found genes that may represent adaptations for iron oxidation, including a variety of extracellular electron uptake mechanisms. FeOB genomes encoded more predictedc-type cytochromes than NOB genomes, notably more multihemec-type cytochromes (MHCs) with >10 CXXCH motifs. These include homologs of several predicted outer membrane porin-MHC complexes, including MtoAB and Uet. MHCs efficiently conduct electrons across longer distances and function across a wide range of redox potentials that overlap with mineral redox potentials, which can expand the range of usable iron substrates. Overall, the results of pangenome analyses suggest that the Gallionellaceae generaGallionella,Sideroxydans, andFerriphaselushave acquired a range of adaptations to succeed in various environments but are primarily iron oxidizers.

    IMPORTANCE

    Neutrophilic iron-oxidizing bacteria (FeOB) produce copious iron (oxyhydr)oxides that can profoundly influence biogeochemical cycles, notably the fate of carbon and many metals. To fully understand environmental microbial iron oxidation, we need a thorough accounting of iron oxidation mechanisms. In this study, we show the Gallionellaceae FeOB genomes encode both characterized iron oxidases as well as uncharacterized multiheme cytochromes (MHCs). MHCs are predicted to transfer electrons from extracellular substrates and likely confer metabolic capabilities that help Gallionellaceae occupy a range of different iron- and mineral-rich niches. Gallionellaceae appear to specialize in iron oxidation, so it would be advantageous for them to have multiple mechanisms to oxidize various forms of iron, given the many iron minerals on Earth, as well as the physiological and kinetic challenges faced by FeOB. The multiple iron/mineral oxidation mechanisms may help drive the widespread ecological success of Gallionellaceae.

     
    more » « less
  4. Abstract Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S 0 )-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S 0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters. 
    more » « less
  5. null (Ed.)
    Ferrimicrobium acidiphilum is a Gram-positive member of the Actinobacteria phylum that can respire aerobically or anaerobically with soluble Fe(II) or Fe(III), respectively, in sulfuric acid at pH 1.5. Cyclic voltammetry measurements using intact F. acidiphilum at pH 1.5 produced fully reversible voltammograms that were highly reproducible. The maximum current observed with the anodic peak was considerably less than was the maximum current observed with the cathodic peak. This difference was attributed to the competition between the platinum electrode and the soluble oxygen for the available electrons that were introduced by the cathodic wave into this facultative aerobic organism. The standard reduction potential of the intact organism was determined to be 786 mV vs. the standard hydrogen electrode, slightly more positive than that of 735 mV that was determined for soluble iron at pH 1.5 using the same apparatus. Chronocoulometry measurements conducted at different cell densities revealed that the intact organism remained in close proximity to the working electrode during the measurement, whereas soluble ionic iron did not. When the cyclic voltammetry of intact F. acidiphilum was monitored using an integrating cavity absorption meter, the only small changes in absorbance that were detected were consistent with the participation of a cellular cytochrome with reduced absorbance peaks at 448 and 605 nm. The cytochrome that participated in the exchange of electrons between the intact organism and extracellular solid electrodes like platinum was the same cytochrome whose oxidation was previously shown to be rate-limiting when the organism respired aerobically on extracellular soluble iron. 
    more » « less