Novel Electron Transfer Systems in Hyperthermophilic Methanogenic and Anaerobic Methanotrophic Archaea: F420-dependent Nitrite Reductase and [Fe-S] Cluster Assembling Thioredoxin

Abstract

Methanogens produce methane as the end-product of their energy respiration, and these microbes can be found in various anaerobic environments. Their metabolisms and unique enzymes have continually expanded the boundaries of our knowledge about microbial energy conservation and opened the potential for bioinspired catalysts. They have also garnered attention as potential microbial cell factories for value-added products. Considering their prospects in science, technology, and engineering, the research presented in this dissertation aims to advance our understanding of the ecophysiology and metabolism of methanogens. Here, we focus on two of their redox enzymes and emphasize the electron transfer functions. From an investigation employing Methanocaldococcus jannaschii, an ancient lineage hyperthermophilic methanogen and inhabitant of deep-sea hydrothermal vents, as a model organism, our laboratory has discovered three novel enzymes linked to the organism's survival strategies, an F420-dependent sulfite reductase (Fsr), deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR), and a thioredoxin (Trx) homolog with a new function. These proteins are involved in redox reactions, and the research presented here deals with the biochemistry of an Fsr and the Trx homolog. M. jannaschii Fsr (MjFsr) performs sulfite (SO32-) detoxification, converting SO32- encountered in its environment to sulfide (S2-), an essential growth nutrient of the organism. In this dissertation, we describe that MjFsr is also proficient in reducing nitrite (NO2-), another toxin harmful to methanogens, to ammonium (NH4+), with hydroxylamine (NH2OH) as an intermediate. Since the mid-point reduction potential of NO2-/NH4+ pair (E0' = +440 mV) is more positive than that of HSO3-/HS- (E0' = ‒116 mV), we rationalize that if reduced coenzyme F420 (F420H2)-derived electrons are proficient for SO32- reduction, they also will be so for NO2- reduction. The enzyme F420-dependent nitrite reductase (FNiR), a homolog of MjFsr, has been identified in anaerobic methanotrophic archaea (ANME), inhabitants of deep-sea methane seeps. This dissertation and the collaborative work show that, unlike MjFsr, FNiR cannot perform sulfite reduction with F420H2 as a reductant and does not confer SO32- resistance to ANME. Yet, it catalyzes the MjFsr-type partial reactions specific to N- and C-terminal domains. It can reduce NO2- and NH2OH. Protein structure modeling suggests that FNiR assembles fewer [Fe4-S4] clusters (4-5 clusters) than MjFsr (5-6 clusters), and chemical analysis results support this prediction. Based on these results, we hypothesize that the observed activity of FNiR, unlike that of Fsr, is likely due to the unique roles of [Fe-S] clusters in connecting the N- and C-terminal domains. Since FNiR carries only four such clusters, the author theorizes that the potential of electrons reaching the oxyanion reduction site in FNiR is too positive for SO32- reduction but negative enough for NO2- reduction. Here, our study has shown a case where reorganization in the reductant processing system could alter the enzyme's redox properties and substrate specificity without changing the active site architecture. The second enzyme studied is M. jannaschii Trx2 (MjTrx2). A Trx system modifies various proteins through dithiol-disulfide exchange reactions, influencing their structures and activities. In M. jannaschii, this system consists of apparently two Trx homologs, MjTrx1 and MjTrx2, a novel Trx reductase utilizing F420H2, termed DFTR, and F420H2 as a reductant. MjTrx1 has disulfide reductase activity, interacts with many proteins essential for growth, and receives electrons from MjDFTR. In contrast, MjTrx2 has poor disulfide reductase activity and does not interact with MjDFTR. Our study shows that MjTrx2 in a homotetrameric form assembles one [Fe4-S4] cluster, a rare occurrence for a Trx. This cluster is sensitive to oxygen, has E0' of ‒386 mV, likely could transfer 2 electrons, and potentially has an oxidation state of 3+, and some of these properties are unusual for a [Fe4-S4] cluster. These characteristics have led to the hypothesis that MjTrx2 could function as a redox sensor. In vivo, the holo and apo forms of the protein, with and without the cluster, respectively, could exist in equilibrium. The author theorizes that during high H2 partial pressures (pH2) (reducing and energy-rich) conditions, holoMjTrx2 predominates and likely facilitates electron transfer. However, during low pH2 (oxidizing and energy-poor) conditions, the protein attains an apo form, losing its ability to perform electron transfer. If proven correct, microbes could use such proteins as a powerful tool to tune their metabolism and better adapt to their environment.