John Peters

A core interest in the laboratory, and an area in which we have made several seminar contributions, is the structure and mechanism of hydrogenases and nitrogenases. Contributed significant structural and mechanistic insights on metal-containing hydrogenases and nitrogenases. Captured and structurally characterized enzymes in multiple mechanistically relevant states by X-ray diffraction methods. This work revealed new paradigms for metal cluster structures and cofactor-based oxidation-reduction catalysis (primarily FeS clusters and flavins), providing inspiration and a framework for numerous studies in laboratories and programs worldwide. Poised states along the pathway of catalysis were captured in crystals, and much of the work involved developing unique tools and methodologies that have had a lasting impact. The results of our work on hydrogenase structure and mechanism have provided the basis for a general hypothesis for effecting catalytic bias in redox enzymes. We have proposed that catalytic bias in any redox enzyme results from the relative stabilization or destabilization of different oxidation states of the enzyme in a system where the rate-limiting step for one direction of the reaction differs from that for the other. Catalytic bias is a current major thrust in the laboratory funded by the National Institutes of Health NIGMS. Our interest in hydrogenase diversity has led us to investigate the mechanism of electron bifurcation, which couples endergonic and exergonic electron-transfer reactions to conserve energy and increase metabolic efficiency. This work is funded by the Department of Energy BES and relates to our interest in nitrogenase structure and mechanism, investigating an electron-bifurcating complex from a nitrogen-fixing microorganism that generates low-potential electron carriers required for nitrogenase catalysis. The results of our work on nitrogenase structure and mechanism have led to studies examining biohybrids of nitrogenase and CdS particles, yielding a suite of seminal findings that address key aspects of the nitrogenase mechanism. The project is a collaboration with DOE National Laboratory of the Rockies and funded by the Department of Energy BES. The system in which electron transfer can be controlled by light has enabled synchronous electron transfer, thereby stabilizing and capturing mechanistic intermediates for the first time, providing new mechanistic insights and advancing our understanding of the nitrogenase mechanism.

Catalytic bias can be defined as the propensity of a catalyst, an enzyme for our particular interest, to accelerate the rate of reaction differently in one direction versus the other direction. We became interested in the phenomenon when we observed that three [FeFe]-hydrogenase orthologs had significantly different reactivity. Most notably, the ratio of hydrogen oxidation to proton reduction rates in these orthologs from Clostridium pasteurianum, termed CpI, CpII, and CpIII, differed by more than seven orders of magnitude. We determined that there were three hydrogenases by sequencing the genome and showed that they were differentially expressed under different physiological conditions. Our hypothesis is that differences in the amino acid composition of the [FeFe]-hydrogenase surrounding the active-site H cluster among CpI, CpII, and CpIII are key determinants of the catalytic bias. We have shown that CpI, CpII, and CpIII differ significantly in the equilibrium of catalytic intermediates as a function of reduction potential. We are testing the hyp othesis using site-specific amino acid substitutions and probing specifically the equilibrium of intermediates using Fourier transform infrared spectroscopy. We have recently begun probing a new model system for catalytic bias. We are using alcohol dehydrogenase as a model specifically because it has been well studied and exhibits an inherent bias toward aldehyde reduction, the reverse of the physiological reaction, alcohol oxidation. We are using site-specific amino acid substitution studies to stabilize or destabilize intermediates and examine the structural determinants and the limits of control over catalytic bias. We are measuring individual rate constants of the interconversion of reaction intermediates using steady-state and presteady kinetics. Structures of variants and captured intermediate states are determined by X-ray crystallography for both our hydrogenase and alcohol dehydrogenase systems; we have obtained structures with resolutions approaching 1 Å.

Electron bifurcation is a term used to describe a phenomenon in which multiple redox-center enzyme complexes modulate the coupling between endergonic and exergonic redox reactions. The concept was first introduced as part of Peter Mitchell’s Chemiosmotic Hypothesis, in which he drew on observations by Marten Wikstrom on the biochemistry of Respiratory Complex III and the role of the “Q cycle” in generating a proton gradient. Flavin-based electron bifurcation was discovered decades later and shares several features in common, largely due to the similar properties of quinones and flavins. There are several examples that have now been reported, and this is an active area where new electron-bifurcating enzymes are being reported frequently.  A large group of flavin electron-bifurcating enzymes couples the oxidation of NADH to that of various metabolic intermediates, thereby reducing electron carriers required for various processes. The process, which conserves energy, is used by a number of anaerobic microorganisms to balance their energetics and is essential for viability. We have made several seminal contributions to understanding the mechanism of these fascinating enzyme complexes. We are focusing on a couple of model enzymes, including Nfn, which catalyzes the NADPH-dependent reduction of NAD⁺ and ferredoxin, and FixABCX, which catalyzes the NADH-dependent reduction of quinone and flavodoxin. We use a variety of direct techniques to examine the properties of numerous redox centers, including UV/Vis, fluorescence, EPR, and protein film electrochemistry. We use steady-state and presteady-state kinetics to study enzyme activities, and we are characterizing structures of intermediates using small-angle X-ray scattering and cryo-electron microscopy. Recent work published in the Proceedings of the National Academy of Sciences USA has been recognized by the prestigious Cozzareilla and Faraday Horizon Prizes.

We have also historically been interested in cofactor and coenzyme biosynthesis and maturation. We have provided significant insights into [FeFe]-hydrogenase H cluster biosynthesis, involving multiple radical SAM enzymes, and into coenzyme M biosynthesis in bacteria, a profound case of convergent evolution.