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1. Ferrimicrobium acidiphilum Exchanges Electrons With a Platinum Electrode via a Cytochrome With Reduced Absorbance Maxima at 448 and 605 nm

   https://doi.org/10.3389/fmicb.2021.705187  

   Blake, Robert C. ; Nautiyal, Amit ; Smith, Kayla A. ; Walton, Noelle N. ; Pendleton, Brealand ; Wang, Zhe ( July 2021 , Frontiers in Microbiology)

   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 cellularmore »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.« less
2. 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

   https://doi.org/10.3389/fmicb.2021.673066  

   Blake, Robert C. ; Shively, John E. ; Timkovich, Russell ; White, Richard Allen ( May 2021 , Frontiers in Microbiology)

   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, whenmore »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.« less
3. Chapter three - In situ absorbance measurements: a new means to study respiratory electron transfer in chemolithotrophic microorganisms

   https://doi.org/10.1016/bs.ampbs.2020.01.003  

   Blake, II ; White, III ( July 2020 , Advances in microbial physiology)

   Absorbance measurements on intact chemolithotrophic microorganisms that respire aerobically on soluble iron are described that used a novel integrating cavity absorption meter to eliminate the effects of light scattering on the experimental results. Steady state kinetic measurements on ferric iron production by intact cells revealed that the Michaelis Menten equation described the initial rates of product formation for at least 8 different chemolithotrophic microorganisms in 6 phyla distributed equally among the archaea and the Gram negative and Gram positive eubacteria. Cell-monitored turnover measurements during aerobic respiration on soluble iron by the same 12 intact microorganisms revealed six different patterns of iron-dependent absorbance changes, suggesting that there may be at least six different sets of prosthetic groups and biomolecules that can accomplish aerobic respiration on soluble iron. Detailed kinetic studies revealed that the 3-component iron respiratory chain of Acidithiobacillus ferrooxidans functioned as an ensemble with a single macroscopic rate constant when the iron-reduced proteins were oxidized in the presence of excess molecular oxygen. The principal member of this 3-component system was a cupredoxin called rusticyanin that was present in the periplasm of At. ferrooxidans at an approximate concentration of 350 mg/mL, an observation that provides new insights into the crowded environmentsmore »in the periplasms of Gram negative eubacteria that conduct electrons across their periplasm. The ability to conduct direct spectrophotometric measurements under noninvasive physiological conditions represents a new and powerful approach to examine the rates and extents of biological events in situ without disrupting the complexity of the live cellular environment.« less
4. Evidence for Quinol Oxidation Activity of ImoA, a Novel NapC/NirT Family Protein from the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1

   https://doi.org/10.1128/mbio.02150-22  

   Jain, Abhiney ; Coelho, Anaísa ; Madjarov, Joana ; Paquete, Catarina M. ; Gralnick, Jeffrey A. ( September 2022 , mBio)

   Newman, Dianne K. (Ed.)

   ABSTRACT Sideroxydans species are important chemolithoautotrophic Fe(II)-oxidizing bacteria in freshwater environments and play a role in biogeochemical cycling of multiple elements. Due to difficulties in laboratory cultivation and genetic intractability, the electron transport proteins required for the growth and survival of this organism remain understudied. In Sideroxydans lithotrophicus ES-1, it is proposed that the Mto pathway transfers electrons from extracellular Fe(II) oxidation across the periplasm to an inner membrane NapC/NirT family protein encoded by Slit_2495 to reduce the quinone pool. Based on sequence similarity, Slit_2495 has been putatively called CymA, a NapC/NirT family protein which in Shewanella oneidensis MR-1 oxidizes the quinol pool during anaerobic respiration of a wide range of substrates. However, our phylogenetic analysis using the alignment of different NapC/NirT family proteins shows that Slit_2495 clusters closer to NirT sequences than to CymA. We propose the name ImoA (inner membrane oxidoreductase) for Slit_2495. Our data demonstrate that ImoA can oxidize quinol pools in the inner membrane and is able to functionally replace CymA in S. oneidensis. The ability of ImoA to oxidize quinol in vivo as opposed to its proposed function of reducing quinone raises questions about the directionality and/or reversibility of electron flow through the Mto pathwaymore »in S. lithotrophicus. IMPORTANCE Fe(II)-oxidizing bacteria play an important role in biogeochemical cycles. At circumneutral pH, these organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside the cell, potentially through a porin-cytochrome complex in the outer membrane encoded by the Mto pathway. Electrons from Fe(II) oxidation would then be transported to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. Directly demonstrating the functionality of genes in neutrophilic iron oxidizers is challenging due to the absence of robust genetic methods. Here, we heterologously expressed a NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to reduce the quinone pool. ImoA functionally replaced the inner membrane c-type cytochrome CymA in the Fe(III)-reducing bacterium Shewanella oneidensis. We suggest that ImoA may function primarily to oxidize quinol inS. lithotrophicus.« less
5. Electrocatalytic Conversion of Furanic Compounds for Production of Valuable Chemicals

   Li, Wenzhen ; Chadderdon, Xiaotong ; Chadderdon, David ; Liu, Hengzhou ( August 2021 , 72nd International Society of Electrochemistry Meeting)

   Biomass is abundant, inexpensive and renewable, therefore, it is highly expected to play a significant role in our future energy and chemical landscapes. The US DOE has identified furanic compounds (furfural and 5-(hydroxymethyl)furfural (HMF)) as the top platform building-block chemicals that can be readily derived from biomass sources [1]. Electrocatalytic conversion of these furanic compounds is an emerging route for the sustainable production of valuable chemicals [2, 3]. In my presentation, I will first present our recent mechanistic study of electrochemical reduction of furfural through a combination of voltammetry, preparative electrolysis, thiol-electrode modifications, and kinetic isotope studies [4]. It is demonstrated that two distinct mechanisms are operable on metallic Cu electrodes in acidic electrolytes: (i) electrocatalytic hydrogenation (ECH) and (ii) direct electroreduction. The contributions of each mechanism to the observed product distribution are clarified by evaluating the requirement for direct chemical interactions with the electrode surface and the role of adsorbed hydrogen. Further analysis reveals that hydrogenation and hydrogenolysis products are generated by parallel ECH pathways. Understanding the underlying mechanisms enables the manipulation of furfural reduction by rationally tuning the electrode potential, electrolyte pH, and furfural concentration to promote selective formation of important bio-based polymer precursors and fuels. Next, Imore »will present electrocatalytic conversion of HMF to two biobased monomers in an H-type electrochemical cell [5]. HMF reduction (hydrogenation) to 2,5-bis(hydroxymethyl)furan (BHMF) was achieved under mild electrolyte conditions and ambient temperature using a Ag/C cathode. Meanwhile, HMF oxidation to 2,5-furandicarboxylic acid (FDCA) with ~100% efficiency was facilitated under the same conditions by a homogeneous nitroxyl radical redox mediator, together with an inexpensive carbon felt anode. The selectivity and efficiency for Ag-catalyzed BHMF formation were sensitive to cathode potential, owing to competition from HMF hydrodimerization reactions and water reduction (hydrogen evolution). Moreover, the carbon support of Ag/C was active for HMF reduction and contributed to undesired dimer/oligomer formation at strongly cathodic potentials. As a result, high BHMF selectivity and efficiency were only achieved within a narrow potential range near –1.3 V. Fortunately, the selectivity of redox-mediated HMF oxidation was insensitive to anode potential, thus allowing HMF hydrogenation and oxidation half reactions to be performed together in a single cathode-potential-controlled cell. Electrocatalytic HMF conversion in a paired cell achieved high molar yields of BHMF and FDCA, and nearly doubled electron efficiency compared to the unpaired cell. Finally, I will briefly introduce our recent work on the development of a three-electrode flow cell with an oxide-derived Ag (OD-Ag) cathode and catbon felt anode for paring elecatalytic oxidation and reduction of HMF. The flow cell has a remarkable cell voltage reduction from ~7.5 V to ~2.0 V by transferring the electrolysis from the H-type cell to the flow cell [6]. This represents a more than fourfold increase in the energy efficiency at the 10 mA operation. A combined faradaic efficiency of 163% was obtained to BHMF and FDCA. Alternatively, the anodic hydrogen oxidation reaction on platinum further reduced the cell voltage to only ~0.85 V at 10 mA operation. These paired processes have shown potential for integrating renewable electricity and carbon for distributed chemical manufacturing in the future.« less