 she has long-standing collaborators here, and her son just graduated last year from UW-Madison with undergrad degrees in philosophy and physics. A rumor received her undergraduate master's degrees in botany from Delhi University in India before moving to the Rensselaer Polytechnic Institute for a PhD in biochemistry. Then as I said, she went to University of Michigan where she was a postdoc with Rowena Matthews before starting her own lab at University of Nebraska in 1991. While at Nebraska, she was promoted to full professor and founded and led the NIH-funded Redox Biology Center. She then moved to University of Michigan in 2007 where she is the Vincent Massey Collegiate Professor of Biological Chemistry. She has a large number of scientific appointments and awards, including the Merck Award from ASBMB and named an ASBMB Fellow, and she's the editor for a number of journals including biochemistry, JBC, and chemical reviews. She is also a member of the Minority Affairs Committee for ASBMB and is the co-director of the ASBMB Mosaic Program, which is a NIH-funded effort to enhance diversity in the academic biomedical workforce. Her scientific accomplishments are numerous, but often focus on the biogenesis and oxidation of hydrogen sulfide, which we'll hear about today, or the enzymes involved in the assimilation, trafficking, and use of vitamin B12. So Ruma is someone who I've known, I guess I would say now virtually for many years. So as a graduate student, her papers were required, a reading for me in my graduate student lab. And I hadn't met Ruma before, but a few years ago, our paths crossed at a study section, and I thought two things. One, Ruma is a reviewer who gives you faith in the academic science review system for the US, that this works, and two, we really need to get her out here to give a talk in a seminar. And so I'm glad that this was able to happen. So with that, let's please welcome Ruma for the 2022 Green Lecture in Enzyme Chemistry. Thank you, Erin, for that very generous introduction, and thank you all for showing up, because we are still operating in this hybrid mode in Michigan, and I have to confess that I've been listening to all our seminars virtually. So thank you for being brave and being here in person. It's a real honor for me to be here as the David Green lecturer. My association with this university and actually with the Enzyme Institute goes back to many decades when I was on my pre-tenure round of giving seminars at various universities. The Enzyme Research Institute was one of the places that hosted me, and of course at that time, in addition to Barry and George, who are in the audience who were present, Mo Cleland was part of this very illustrious group of enzymologists who continued the tradition that was started by David Green. So as you have heard, David Green was the founder, of course, of your Enzyme Research Institute here. He was the father of Rowena Matthews, who was my postdoc mentor, and this was taken just the summer before the COVID lockdown and that's Rowena and her husband Larry, who unfortunately passed away in November of last year. So I also feel connected, obviously, to this lineage. So academically that's my lineage. Recently as I was, oh yeah, actually just last week I was at the ACS meeting in San Diego and was one of the speakers at the 75th anniversary of the Pfizer symposium. So the Pfizer award has been given traditionally to enzymologists and I discovered at that symposium that David Green was the winner of the first Pfizer award and I won this in 2001. So there was another connection that I discovered just last week. So in reviewing some of the many, many contributions that David Green made to biochemistry, I decided to highlight just a few. And the first is this, he was obviously very audacious in the way he thought, in the way he impacted science and at the age of 30 he wrote this book on the mechanisms of biological oxidations which basically covered redox reactions that were catalyzed by enzyme and a book that was very influential for a long time. And a year later he published his musings on trace substances. So these were both metals and cofactors and he postulated that their importance in biological systems was most likely to be through their properties as cofactors of enzymes. And finally during his time here he oversaw but was not co-author on this paper but this was an important paper in which coenzyme Q was functionally connected to complexes one, two and three as the mobile redox cofactor that connected these three complexes. So what I decided to do today is to really focus on a story from my own lab that intersects in all these ways with David Green's own work and it's a story that resides in the mitochondrion and it involves this very small molecule hydrogen sulfide. So historically hydrogen sulfide has been known as an environmental toxin associated with areas of geothermal activity. It's also what gives rotten eggs their putrid odor. But actually the emergence of life on this planet is also intimately connected with hydrogen sulfide chemical biology because it's been posited that as the sulfide-laden alkaline waters from hydrothermal vents mixed with the acidic waters of the Hadean Sea that iron sulfa colloidal iron sulfa mats formed and these catalyze the synthesis of the first organic molecules. But over the last 20 years or so, hydrogen sulfide has been increasingly associated with modulating a large number of physiological effects as it's been recognized to be a product of our own metabolism. And so the question is, why do we make H2S if it's a toxic molecule and how does it influence so many different physiological effects? So just to illustrate a couple of examples here, we've all heard of the cardioprotective effects of garlic. This is not just an old wives' tale. It turns out that garlic is chock full of allele sulfide which upon ingestion release hydrogen sulfide which like nitric oxide leads to vasodilation. And at the other end of its biological spectrum is it's a potent inhibition of respiration by virtue of targeting this bimetallic center in complex four and as it does so, it has a profound impact on metabolism because it is able to slow metabolism in a reversible manner inducing a state of suspended animation or hibernation even in non-hybernating animals like a lab mouse. So we're really interested in my lab in understanding how hydrogen sulfide signals and specifically on how it impacts metabolism. So how is H2S made? Hydrogen sulfide is a product of sulfur metabolism. It's derived from these amino acid cysteine and homocysteine and two of the three enzymes that catalyze H2S synthesis are enzymes that belong to the transulfuration pathway and their predominant H2S releasing reactions are shown here and they're basically classic PLP dependent elimination reactions that eliminate HS minus or H2S from cysteine and or homocysteine. The third enzyme is a sulfur transferase macapto pyruvate sulfur transferase and as its name suggests, it transfers sulfane sulfur to an acceptor and eventually hydrogen sulfide can be released from there. Okay, so because we make hydrogen sulfide and it's toxic we also have a pathway that is dedicated to its oxidation and this pathway resides in the mitochondrion and it begins with this diflavin oxidoreductase sulfide quinone oxidoreductase that oxidizes H2S to upper sulfide and it slips the two electrons to coenzyme Q which then enter the electron transport chain at the level of complex three. So this makes hydrogen sulfide the first known inorganic substrate for the electron transport chain that's known in mammalian cells. So the rest of the pathway is basically a series of somersaults through oxygenases and sulfur transferase reactions and these lead to the elimination of either thiosulfate or sulfate as the end product of sulfide oxidation. So what about signaling? How does hydrogen sulfide signal? And so the predominant view in the field is that it signals via this post translational modification called persulfidation and we and others have shown that these post translational marks definitely exist in the cell and in fact there are hundreds of proteins that have been identified that have persulfide modifications and their number only increases in cells that have been exposed to H2S. But what I'd like to point out is that we have yet to see an example of a quantitatively significant of a system in which the persulfidation levels are quantitatively significant and that has been associated and that significant modification level has been associated or clearly demonstrated to be functionally relevant. So while these post translational markings definitely exist in the proteome, their relevance to function is certainly very much of an open question. So in my lab we have focused instead on the effects of H2S on redox remodeling and that by that I mean metabolic remodeling focusing on the electron transport chain where the impact of H2S has long been known. So that this is a bona fide target of H2S and as you all know the electron transport chain is a major currency converter in cells where the redox status of these important cofactors like flavin, pyridine nucleotides and quinones, so their redox spoils is very intimately controlled by the activity of the electron transport chain. So the two vignettes that I would like to share with you today focus on first the unusual redox enzymology of this enzyme which is a sulfide, quinone, oxido reductase that catalyzes the committing step in the conversion of hydrogen sulfide to persulfide. And second I would like to share this recent discovery in our lab that the electron transport chain is forked and that in addition to its operation in this canonical manner where oxygen gets churned to water at complex four, it can also bifurcate at the level of CoQH2 and move electrons to fumarate as a terminal electron acceptor converting fumarate to succinate. Okay, so let's begin with sulfide, quinone, oxido reductase. This is as I mentioned a flavin enzyme and it belongs to the family of flavin disulfide, oxido reductase that are characterized by a flavin cofactor and a cysteine disulfide. So that's the redox motif in the super family of enzymes. And so the predicted reaction mechanism for SQOR was that sulfide adds into this disulfide, forms a persulfide and a charge transfer complex. And then in the next step, this outer sulfane sulfur is moved to a thiophilic acceptor like glutathione to generate the persulfide. So once the sulfide oxidation step is complete, the rest of the half reaction basically is a redox reaction in which electrons are moved to the flavin and then out of the enzyme through this mobile electron acceptor coenzyme Q. So a lot of the work that I'm going to be describing today was done by a very talented postdoc Aaron in my lab who actually was the last day of his tenure in my lab was last Friday. He's now moved on to a chemical company. Okay, so a couple of years ago now, there were two groups that basically in the same year published the first structure of human SQOR. The structure in my lab was solved by another postdoc, Sojin Moon. And so this is the structure of human SQOR with the two red helices indicating the two helices that anchor this protein into the mitochondrial membrane. And what you can see out here is that we had crystallized the enzyme with flavin. What was the surprise in this structure was that there was an extra electron density between the two cysteines and using sulfur anomalous diffraction data, we were able to assign this extra sulfur or confirm the assignment of this extra sulfur as extra density as a sulfur. So we concluded that the resting form of the enzyme was actually a trisulfide rather than a disulfide, a trisulfide that had never been previously seen. So while Marilyn Jorns' lab concluded that the trisulfide was an artifact of the isolation process, we went on and obtained many more structures of human SQOR. So the next one that is shown here was obtained in the presence of CoQ. And we saw a nice electron density of CoQ in the structure proximal to the flavin and so really positioned to accept electrons from the flavin. We also saw that in this particular structure, this trisulfide was opened and what we were seeing here was density that was consistent with the presence of a bisper sulfide. So in addition, this enzyme SQOR is actually pretty promiscuous and it can transfer that sulfane sulfur from the bisper sulfide to a variety of acceptors. And one of the acceptors that works is hydrogen sulfide. And so when we diffused hydrogen sulfide into these crystals, what we found was completion of catalysis. So what was really cool was that the trisulfide reformed and all this happened in crystal low. And we also found that the electron density for CoQ had moved far away from the flavin and was now actually, we interpreted this as evidence that CoQ was caught diffusing out of the active site in its reduced form. So based on this series of crystallographic snapshots, what we concluded was the active form of this enzyme is actually the trisulfide rather than the disulfide. And to further confirm this assignment, we did a biochemical analysis of the sulfane sulfur that is associated with this enzyme as isolated and at the end of a single catalytic turnover. And so called cyanolysis, this is pretty classic chemistry to look for sulfane sulfur. So called cyanolysis basically detected the presence of a single extra, a single sulfane sulfur, both at the beginning and end of turnover, which is consistent with the model that this trisulfide is in fact the active form of the enzyme or the resting form of the enzyme. So this then led us to postulate a new mechanism for SQOR which begins with the trisulfide and upon addition of the sulfide results in the formation of a bisper sulfide which is involved in the charge transfer complex. The charge transfer complex that we and others had recorded for this enzyme is unusually intense and before we had the structure of this charge transfer complex, we had speculated that electronically there was something different about it. And so I think the something different is now suggested by the structure as well as now the postulated mechanism which is this electron extra electron density from having two persulfides in the vicinity of the flavin. The rest of the mechanism is basically the same. You move the sulfane sulfur out of here to the acceptor, move electrons into the flavin and then out through CoQ. So of course we asked the question trisulfide, what is the catalytic advantage of having a trisulfide? I mean the rest of this super family has an active side sulfide and to address this question we collaborated with Laura's lab in Uruguay and using DFT PCM modeling, she showed that this is the transition, calculated transition state for sulfide attacking a trisulfide versus a disulfide that the activation energy is significantly lower for achieving this transition state versus when you start with a disulfide. And that basically translates to a 10 to the fifth fold rate enhancement that a trisulfide cofactor offers over a disulfide cofactor. Okay, so getting a little bit into the biology of this enzyme and its role in the mitochondrion. As I've mentioned, H2S is a substrate for the electron transport chain and as it is utilized by SQOR it stimulates oxygen consumption as electrons move down the ETC. But as levels of H2S increase it also becomes an inhibitor. So there's really a window within which hydrogen sulfide acts as a substrate and also acts as and then acts as a respiratory poison. And so we asked, we looked at this OCR stands for oxygen consumption rate and this looks a little bit complicated but it's really a kinetic trace of oxygen consumption by cells that have been exposed to a variety of different concentrations of H2S. So you see that the basal rate spikes up as concentrations of H2S up to 30 micromolar are added to the cell suspension. But somewhere between 30 and 40 micromolar there's net inhibition of oxygen consumption and that's because there is a net inhibition of respiration at that concentration for these cells. Okay, so what happens when we take out SQOR and so we knocked it down using SHRNA in a couple of different cell lines and what we found was that these cells now at every single concentration of H2S and we've gone down to one, two and five micromolar as well these cells basically keel over and they are poisoned at all the concentrations of H2S that we have examined. So based on this data we can conclude that sulfide quinone oxidoreductase is a respiratory shield and its presence is important for protecting against respiratory poisoning. Also a couple of years ago in collaboration with clinical colleagues we described the first hereditary mutations in human SQOR and these were described in two siblings who succumbed at the ages of four and eight years. These were the clinical presentations and basically they presented a lot like Lee disease which is this polygenic disease that results from mitochondrial dysfunction. So the missense mutation that was inherited by both these siblings was a glutamate to lysine substitution and at the time that this was mapped we had just obtained the structure of human SQOR and in fact the structure predicted that the substitution of glutamate to an, by an arginine here would be very destabilizing and in fact in the very tiny amounts of protein that we obtained, tissue that we obtained from these patients we were able to show that that in fact was the problem. So to summarize what I have shown you so far SQOR has a novel trisulfide redox motif which enhances the rate of H2S addition by a factor of 10 to the fifth that SQOR functions as a respiratory shield and that mutations are inherited as autosomal recessive disease and they present as Lee's disease. And so moving on then I'm going to tell you the story of the electron transport chain and as I've already mentioned our discovery that this electron transport chain is in fact forked. So just to step back here for a moment most of our work on hydrogen sulfide is focused on cells that line the colon. So the colon epithelial cells are routinely exposed to high levels of H2S which are a product of microbial metabolism. So this is a system which allows us to interrogate this interaction between diet, microbial and host metabolism and we use in addition to the type of in vitro studies that I've just shown we also use cell culture and mouse models to interrogate the chemical biology of H2S signaling. So this is just a heat map showing that in H2S treated cells versus control cells there are a very large number of metabolites that either go up or go down and this somewhat busy scheme here is simply to remind me to tell you that the sulfide oxidation pathway that's shown in blue intersects with the electron transport chain and changes here have the potential to move outside the mitochondrion through metabolic networks like the malatis partate shuttle which communicate redox status in an intercomport mental manner. Okay, so one of our first observations with H2S was that it stimulates aerobic glycolysis and what is aerobic glycolysis? It's basically the dumping of glucose carbons as lactate and the rationale for doing this when the electron transport chain is blocked which is what you expect with H2S is that it sets up a redox neutral cycle in which NADH that is generated by glycolysis is recycled to NAD by lactate dehydrogenase and this becomes really important if you want to make ATP in a cell where respiration has been blocked as in H2S treated cells and so we went on to show that increased glucose consumption and lactate production was seen across a variety of cell lines that we had examined and then to ask whether it was the mitochondrial or the cytoplasmic NADH pool that was setting up aerobic glycolysis in H2S treated cells we used this LB NOx which is NADH oxidase so it's a genetically encoded tool which was actually developed by a former graduate student of mine when he was post-talking in Vamsi Mutha's lab and it oxidizes so it dissipates the NADH pool but instead of generating superoxide or peroxide as many of these NADH oxidases do it generates an innocuous product like water so you're really not perturbing the redox status other than the NADH-NAD ratio okay so we can express this either in the cytoplasm or in the mitochondrion and we found that when we expressed LB NOx in the cytoplasm we still saw an increase in glucose consumption but that was not observed when we expressed this in the mitochondrion and we also went on to show that both in a malignant as well as a non-malignant cell line the NAD to NADH ratio went down in H2S treated cells indicating that the cells were experiencing what is called a reductive stress which is much less described in the literature than oxidative stress okay so a classic signature of reductive stress is that it reprograms the Krebs cycle so we're all used to thinking about the Krebs cycle as an oxidative cycle that operates in this direction generates NADH and FADH2 but it's known that when the NAD pool has become reductively shifted you really can't operate this either in the oxidative direction and so this backs up isocytrate dehydrogenase which uses NADPH catalyzes the reductive carboxylation of alpha-ketoglutarate and that's converted ultimately to citrate which becomes a source of carbon that's used for macromolecular synthesis so one way to trace this metabolic shift is by using C13 labeled glutamine and if the citrate is being generated in this reductive pathway you would expect the retention of all five carbons here in citrate whereas if you had citrate being generated in the oxidative direction because of the decarboxylation reactions that are involved here you would see that the citrate would... the M++5 isotopalog would get depleted so what we found was that in H2S treated cells the M++5 pool of citrate increased and that was consistent with a reversal of the Krebs cycle in H2S treated cells so the other thing that we looked for is what happened to the lipid pools in H2S treated cells and so using a very similar labeling strategy starting from glutamine because you really can't label with glucose every glucose carbon is getting dumped as lactate in H2S treated cells so the carbon for biomass synthesis is really coming from other sources like glutamine so what we found was that in H2S treated cells there was a very significant increase in lipid biogenesis and again this was insensitive to the dissipation of the NADH pool in the cytoplasm but sensitive to its dissipation in the mitochondrion and we also did a fairly extensive lipidomics analysis to look at what pools of lipids were changing in H2S treated cells at various time points and some of the changes that we saw across all time points was that the triacylglycerides were up-regulated but the phosphatidylcholine pool was down-regulated so what we had noticed a few years ago when we were working with H2S oxidation and this was work done by a long time a senior associate in my lab, Victor Wittwitzke who retired last fall so Victor had noticed that when he took kidney, brain or liver lysate and exposed them to H2S in an anaerobic chamber that's about as anaerobic as we are able to get experimentally, we continued to see H2S oxidation and that was really puzzling because as I've just shown you H2S oxidation is oxygen dependent at least that's the way we understood it so the question was what was going on here? How did H2S continue to get removed by these tissue lysates? And so here again, this is what we were expecting that as H2S got oxidized, oxygen would be consumed so this told us that there must be some other way in which this oxidation is being supported so this project was picked up by a really talented post-doc in my lab, Roshan who examined this metabolomics data and found that in H2S treated cells there was a very significant accumulation of succinate and some other carbon intermediates including hypersanthine and inocene which as I will show you in a minute connect to the purinucleotide cycle. So what Roshan postulated was that in cells where complex four is inhibited, complex two reverses. So complex two reverses by using fumarate as an electron acceptor and generating succinate which accumulates the rationale for this is that it sets up a redox cycle in which CoQ H2 that is generated via oxidation of H2S is now oxidized and you can continue to prioritize H2S oxidation via establishment of this redox cycle. So you with me with this because this is really fundamentally new that here we're saying that you're oxidizing via the electron transport chain but you're using fumarate rather than oxygen which is what's described in all of our biochemistry textbooks as the ultimate electron acceptor. So to address this experimentally and this was actually addressed many, many different ways I'm gonna show you just a couple of pieces of data. What Roshan did was he took out the catalytic subunit of complex two in a couple of different cells and cell lines and he showed that in both cases H2S consumption went down. So the efficiency went down. He also treated cells with membrane soluble precursor of fumarate dimethyl fumarate and he found that the efficiency of H2S consumed went up. And then using metabolic labeling approaches he showed that the fumarate that is used to support this new redox cycle is sourced from a couple of different metabolic pathways including the purine nucleotide cycle explaining this increase as well as the malatis partate shuttle. So that's there's a significant amount of metabolic rewiring that's going on to support this new activity, this non-canonical activity in the electron transport chain. But what about complex one? There was a literature that indicated that complex one can also reverse transfer electrons in the reverse direction. So complex one activity can also undergo reversal and here the electron acceptor is still oxygen though. So it didn't really fit with our model but we still had to examine it because it was out there in the literature. So if complex one actually sets up this redox cycle with SQOR you would predict that if you took out complex one H2S oxidation would become less efficient. On the other hand, if complex one was simply a competitor and electrons were flowing in the forward direction from complex one, if you took out complex one then H2S oxidation would become more efficient because there would be less competition for the CoQ pool. So what we saw in a couple of different ways was that if we took out complex one genetically H2S consumption increased in efficiency. So that's compatible with a competitive model. It's really not compatible with this model with reverse electron transfer. And if you just focus on this data if we blocked complex one with rotinone again the efficiency of H2S oxidation increased. So these pieces of data basically allowed us to rule out the involvement of complex one in supporting oxygen independent oxidation of H2S. So everything that I have shown you so far was done across multiple cell lines but we asked then what about the physiological relevance? Does this actually happen at an organismal level? And luckily for Roshan he found that there was a lab just down the corridor that had an SDHA floxed mouse so you could take it out, you could take out SDHA in a tissue of interest and the mice that were being bred in this particular lab it was amazing because they had this SDHA knocked out in the intestinal epithelial cells which is exactly where we focus our biological studies on. So the setup of this experiment is that if you take out complex two then H2S oxidation is going to become less efficient and here we are not modulating oxygen, we're not even exposing the mice to H2S. So this is just endogenous H2S metabolism that we are interrogating in the organism. So H2S of course is very difficult to measure and what we measure instead is thiosulfate as a readout for the volume of H2S that is getting oxidized. So the flox of H2S through the oxidative pathway and initially what Roshan looked at was the fecal matter which really reports on colon metabolism and he found that in these SDHA knockout mice there was a very significant decrease in thiosulfate but what was really surprising and this is something that we're following up on was that in these mice there was a systemically significant decrease in thiosulfate as well. So this is in serum as well as in urine indicating that gut H2S metabolism is quantitatively a very significant contributor to systemic H2S metabolism. Okay, so we were very excited about this work because we realized that we had made a fundamental discovery about the mechanism by which the electron transport chain functions and how electrons can flow either forward to onto complex four or complex two can reverse course and use fumarate. This work was published in early November in JBC and two weeks later we found the same discovery was reported in science by Sabatini's group and I should point out that this journal had actually declined to review our manuscript but this discovery was, they came upon exactly the same discovery but using a different route so remember that we were poisoning complex four, we were poisoning a respiration but what they were doing was using hypoxia and they were following pyrimidine synthesis so dihydroorotate dehydrogenase like SQOR is another enzyme that uses CoQ as an electron acceptor and they found that in hypoxic conditions when oxygen becomes limiting and forward electron transport is restricted, fumarate becomes an alternate electron acceptor and in very elegant metabolic labeling studies what they went on to show was that in some tissues like liver, kidney, brain, this was constitutively, fumarate was constitutively being used so these are not hypoxically challenged animals so these are just normal mice that constitutively use fumarate as an alternate electron acceptor, in fact they use fumarate more than they use succinate so there's evidence that complex two is working more frequently in the reverse direction and the forward direction in these three tissues whereas other tissues like the ones that are shown here they scarcely if ever use fumarate as an alternate electron acceptor but they showed that under exercise stress tissues like white adipose tissue and heart they switch metabolism and start using fumarate instead of, I mean oxygen becomes limited under exercise stress. Okay so in the literature what people have argued is that under hypoxic conditions forward electron transport is going to become limited and the reason that they have, the reason for this argument is that although we do a lot of our experiments at 21% oxygen, tissues are actually exposed to much lower concentrations of oxygen and they can be as low as 0.4% in colon to up to 13% in some regions of lung but even within a single organ like kidney the oxygen exposure to cells can range from two to 9%. So this seems like a reasonable way to understand, to rationalize why oxygen might become limiting but of course as an entomologist you want to look at what is the KM for oxygen for complex four and as I looked in the literature it looks like this enzyme is not going to be sensitive to these levels of oxygen fluctuations so within this biological range if the KM for complex four, oxygen for complex four is 0.02, 0.1%. So what we have postulated instead is that gases like H2S and nitric oxide by modulating the affinity of a complex four for oxygen is modulating the flow of electrons through the ETC and of course both H2S as well as nitric oxide are products of our own metabolism. So is there a connection between hypoxia and nitric oxide and H2S? The answer is yes, we can metabolically trace this. Under hypoxia there is a massive program of regulation that is elicited through this hypoxia inducible factor which upregulates the enzymes that make both CO as well as nitric oxide so nitric oxide synthase and heme oxygenase and we had shown many years ago that both these gases are able to inhibit one of the two enzymes in the transulfuration pathway which is a heme protein. So they bind the heme and they shut down synthesis of either cystothinine or H2S by cystothinine beta synthase. So under these conditions what we had postulated and then went on to show was that under these conditions when CBS is blocked the transulfuration pathway switches metabolic track because it's no longer producing cystothinine that's going to be competitive with this next enzyme in the pathway. Cystothinine would make cysteine but because this is now not being made by CBS this enzyme now starts making H2S. And in fact under hypoxic conditions we have recently gone on to show that H2S synthesis is upregulated and we believe that this is one mechanism by which this is possible. Okay so finally why complex two because there are many users of coenzyme Q and some of them are listed here. So complex one, two, a lot of these enzymes why is it that complex two out of all these co-Q users reverses? And I think the answer is very clearly in the redox potential. So if you look at the redox potential for the co-Q, co-Q H2 couple that's what it's reported to be. And if you look at the redox potential for complex two it's very similar so these are sort of isopotential. What about the redox potentials for all these other co-Q users? They're totally mismatched, they're totally at a range. And so it's really only complex two which is poised to couple with co-Q H2 and reverse scores when the conditions are right i.e. co-Q H2 is up and fumarate is available to accept electrons. So I think this is really, it's a design principle within the electron transport chain that allows the alternate use of fumarate as an electron acceptor. Okay, so to summarize then what I have shown you here is that hydrogen sulfide regulates electron flux in the ETC and it can promote reversal at complex two. It has a large number of metabolic effects these metabolic effects start by a redox shift in the reductive direction in the mitochondrion and then this sort of ripples out into the cytoplasm metabolic, yeah this is what I just said and the intestinal epithelium is a very significant contributor to systemic sulfide metabolism by mechanisms that we don't quite understand. And so these are the people in my group that I would like to thank and the funding agencies and for those of you who are young in the audience I have a few more slides that really talks about my journey as a scientist because science just like anything else is so much about the journey as well. So mine started here in Calcutta in India. I was born as an Army brat and at the age of four my father was transferred from here to the south and so the next I guess 10 years was a constant a war of changing places, faces, languages as well in India until I finally graduated from high school and ended up, whoops, ended up in Delhi where I went to university and for reasons I won't get into but my first two degrees were both in botany so I got my BS and MS in botany and then as I was getting close to graduation what I really wanted to study was biochemistry but biochemistry was not a discipline it didn't exist as a discipline in India at that time you either studied chemistry or zoology or botany but it was a chance meeting I was a visit by Joyce Diwan who was a professor at RPI she visited my department at Delhi University in fact she handed me a Manila envelope with the application package and that's how I ended up doing this 10,000 mile journey to upstate New York and it was really a sea change in so many ways because I went from a very large public institution to a very small private institution but in addition to that sort of environmental change I also went from botany and trained with medicinal chemists and learned how to synthesize small molecules I'm almost certain that I was the only person in Jim's very tough lab who was given this opportunity to a switch from biology to a synthetic organic chemistry Jim was very difficult as a mentor as a PhD mentor but he's a very, very fine person and continues to be a good friend so when I finished my PhD in four years I was looking for a postdoc actually the only place that I applied to was the University of Michigan at that time and I was tasked to clone a gene it was actually for a B12 dependent enzyme methionine synthase and manually sequenced 0.1% of the E. coli genome it might seem trivial today but it was very challenging for me because not only because this seemed like a lot of work but my only research experience was in synthetic organic chemistry but Rowena Matthews who was my mentor was constantly encouraging although she was not in the lab herself she was on sabbatical leave during that first year but I finally got this gene and got the protein expressed and had buckets of this beautiful pink enzyme and then spent hours trying to figure out the kinetics of this reaction mechanism shooting enzyme with Dave Ballew and doing hours and hours of stopped flow studies on an instrument that he had made as a graduate student and at that time he was the only person who could really operate that instrument and then I also was interested or actually Rowena was really interested in getting the redox potential of this very low potential cobalt center in B12 and shipped me off to do these EPR spectral electrochemical studies and Steve Ragsdale's lab he was at Milwaukee at that time and these experiments led to an unexpected affluence of both my personal and professional interests and so a couple of years later when I was done with my postdoc training I found myself looking we found ourselves looking not for one but two jobs and ended up in Nebraska and in the middle of cornfields and in Nebraska I had our first child within I think it was four weeks of starting as an assistant professor and he who was at, who took the scenic route and graduated just a few years ago from Wisconsin, he came three years later but Nebraska was actually very challenging the early years were very challenging it was a very different community as soon as I stepped out of the university people would ask me which church I went to before they knew what my name was and so to deal with this irritation I started telling them that I belong to the church of Cobolamine because my lab was Cobolamine is a chemical name of B-12 and somehow that seemed to appease their curiosity and then they left me alone so when I started my career I heeded the advice of Helmut Beiner at some of you who've been here for a little while know who he is he was a really famous entomologist and spectroscopist and was actually at the Enzyme Institute so I remember very clearly I was at a dinner that I think Steve was hosting and Helmut and Jeremy Knowles were there and Helmut was telling me I hadn't started my lab yet that pick a problem that isn't moving and move it and to me that seemed like really, really a sound advice and I can show you, I can tell you that you know the work on hydrogen sulfide which I've been working on sulfa metabolism for a while might for a long time seemed obscure this field is sort of beginning to heat up but our following on the trail of hydrogen sulfide and signaling and not going where 99.9% of the field is which is persulfidation based signaling has led us to a fundamental discovery about how the electron transport chain functions and to redox signaling via reductive stress in the mitochondrion and so it's really been my experience that insights into biology have really repeatedly opened up as we have pursued interesting questions in obscure pathways and you've got to hear one of these stories today I also work on B12 enzymes and B12 trafficking proteins and that's really not would not be considered to be the hotbed of biochemical activity so finally I wanted to leave with just a few words on a program that I have been developing which is an NIH funded mosaic program that is embedded in the ASBMB these are the faces of the scholars they're all K99 awardees and they're all headed to R1 research institutions the goal is to diversify STEM so these are biochemists, chemists, engineers and our goal is really to provide so these are their names some of them have already transitioned as you can see from their postdoc to their faculty positions and many of the others are actually in the midst of negotiating offers right now so our goal is to provide a suite of career development opportunities through ASBMB to enhance our professional networks through the national meeting as well as the journals and enhance institutional accountability so for those of you who are going to be at the ASBMB next week or later this week actually I'm going to be there so those of you who will be there I would invite you to show up at a panel that these scholars are going to be hosting on a race and mental health in STEM so with that I think I'm at the end of my story I'm sorry if I've gone a little long but thank you very much for your attention Thank you Ruma, that was really phenomenal and law inspiring I personally was thinking of like so many questions that are going to be on my undergrad class's final exam next year so maybe I'll just start off with maybe the first question then we can open it up so I was trying to connect the hydrogen sulfide at the end with something you said at the very very beginning about the presence of this gas and like deep thermal vents and so on and I was thinking of you know archaebacteria, the things that look there and I was wondering you know the mammalian system does this speak to either how or the evolutionary origin of mitochondria or potentially microbiome connections that's a great question to which I have no coherent answer so I mean hydrogen sulfide is clearly used as a source of energy by bacteria I don't think it's a significant source of ATP in our cells so I really think that we use it as a regulator and you know time will tell whether our postulate of whether H2S and NO are modulating ETC activity time will tell whether that's true or not but I don't believe that hypoxia modulates it just based on kinetic parameters right but I guess I'm wondering if what we're seeing yeah I know just being used in one of our systems originated in a completely different purpose totally and that seems very reasonable but I don't know that I could comment beyond that yeah any other questions yeah yeah now that was such a phenomenal talk so the first question I have is other models of reductive stress that really focus on reductive stress in like the cytokosm and the effects from like the hemisposphate pathway and specifically how it relates to aggregate diseases and so I'm just curious do you think that H2S has a function solely in the wet climate or do you think it also plays a role in the cytokosm? I mean it clearly has a role beyond the mitochondrion because aerobic glycolysis is another hallmark of reductive stress and so is lipid biogenesis that's glutamine and reductive carboxylation dependant so clearly those things are happening in the cytoplasm whether we've looked at pentose phosphate pathway and we don't see that H2S modulates it at least under the conditions we've looked at and in the cell lines we're very interested in going beyond the cytoplasm though into the ER and potentially into the nucleus as well One more question In terms of the lipid changes so you saw increases in triglycerides and decreases in alcohol PC Do you know at all how much regulated is it through mitochondrial functions? Definitely and I went a little bit quickly over that data but it's only the mitochondrial NADH or NADPH because we have TPNOx that does the same thing but takes out NADPH so if we perturb the NADH or NADPH pools in the mitochondrion we interfere with incorporation of glutamine into lipids and I just don't understand that I was looking forward to talking to lipid experts because after all that synthesis happens in the cytoplasm so how is it that with depleting NADPH in the cytoplasm there's no effect on lipid biogenesis so I don't understand it maybe there's a rush of reducing equivalence that's coming out of the mitochondrion and that's what keeps it going Great That's exactly what you're talking to Yeah, I know So a student in my lab studies the regulation and expression of NADPH Which one? The two or one, two, three? This is in yeast which has three of them but they have CDS domains that are not part of the enzymatic domain and that are that have to be a regulatory domain and I noticed you were seeing the effects in some of your experiments that were modulating and it's self-righted you saw changes in curing nucleotide metabolism Do you think it's possible that hydrogen sulfide could be involved through those CDS in regulation? So the CPS domains for the audience these are domains that actually were found in systethyne beta synthase which is involved in H2S synthesis but now found in lots of other proteins they tend to be important in energy sensing and so I don't think that H2S directly has any impact on CPS domains but they typically bind adenosine derivatives and our protein it binds as adenosyl methionine so ATP levels fall in H2S treated cells which probably is one reason why aerobic glycolysis is triggered so I think if there are changes in the energy status depending on what IMPDH I don't remember I should know this I don't remember what IMPDH responds to which of those nucleotides is it AMP or it actually binds both guanine and adenine nucleotides from phosphorylation states yeah so indirectly it would be impacted but I think the involvement of IMPDH here is really the NEDPH levels are up and that's driving reverse the reductive carboxylation we have time for one last question Judith I'd like to agree totally that this is a phenomenal sum of things I'm curious and my question is very much like what Erin was asking in terms of the evolution and the breadth of this you've worked on in Tesla and Thelia and mammals and do you have to start to explore what other organisms have this kind of port EI chain I'm curious about the evolution clearly it's really good and can you knock out the SQOR and does the cell live I didn't mention that curious so in response to your first question of course it's known that bacteria use all kinds of alternate electron acceptors so the news here is that mammalian cells do too so nitrate fumarate all of these are known tetrothionate these are all known acceptors so I think the agility of bacterial electron transport systems to utilize available electron acceptors is well documented it's just the surprises that the mammalian one is also nifty yeah I mean this is this came out in November these two papers came out in November so I would imagine that we actually focus on mammalian cells no no idea no idea your second question was about so in cells there's you know we can pick up a phenotype in terms of sensitivity to poisoning we now have a phloxed SQOR mouse which means that we can do a whole body SQOR knock down and their problems there with survival but it's not lethal they just don't survive very long we don't know why but what we're doing is really taking it out in a tissue specific way and that's a lot easier to manage that doesn't seem to be so far unless you stress with disease one model that we're interested in is ulcerative colitis you know we're beginning to see phenotypes there but if you take it out in a tissue specific manner it seems to be tolerated but you know we have the clinical data on the patients so it's not lethal so four years eight years and then they succumbed it is null it's autosomal recessive so it's not null you know there was some expression but it was right yeah simple if you know how to work with yeast we don't let's thank Ruma for the fantastic seminar thank you and I really like how you also talked about David Green at the beginning and your journey at the end