Yeast Flavohemoglobin, a Nitric Oxide Oxidoreductase, Is Located in Both the Cytosol and the Mitochondrial Matrix

Abstract

Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in both the oxidative and nitrosative stress responses in Saccharomyces cerevisiae. Previous studies have shown that the expression of YHB1 is optimal under normoxic or hyperoxic conditions, yet respiring yeast cells have low levels of reduced YHb pigment as detected by carbon monoxide (CO) photolysis difference spectroscopy of glucose-reduced cells. Here, we have addressed this apparent discrepancy by determining the intracellular location of the YHb protein and analyzing the relationships between respiration, YHb level, and intracellular location. We have found that although intact respiration-proficient cells lack a YHb CO spectral signature, cell extracts from these cells have both a YHb CO spectral signature and nitric oxide (NO) consuming activity. This suggests either that YHb cannot be reduced in vivo or that YHb heme is maintained in an oxidized state in respiring cells. By using an anti-YHb antibody and CO difference spectroscopy and by measuring NO consumption, we have found that YHb localizes to two distinct intracellular compartments in respiring cells, the mitochondrial matrix and the cytosol. Moreover, we have found that the distribution of YHb between these two compartments is affected by the presence or absence of oxygen and by the mitochondrial genome. The findings suggest that YHb functions in oxidative stress indirectly by consuming NO, which inhibits mitochondrial respiration and leads to enhanced production of reactive oxygen species, and that cells can regulate intracellular distribution of YHb in accordance with this function.

Extension of chronological life span by reduced TOR signaling requires down-regulation of Sch9p and involves increased mitochondrial OXPHOS complex density

“…These results might suggest a role for NO and/or other reactive nitrogen species as relevant. Interesting in this regard, as is the case in tor1 Δ cells (Table 1), Yhb1p localizes to mitochondria under anaerobic condition [37]. This, coupled to our observation that hypoxic conditions bypass the extension of CLS by TOR1 deletion [18] might suggest that reduced TOR signaling and anaerobic conditions share a common route to impact life span that may involve NO metabolism.…”

Identification of Potential Calorie Restriction-Mimicking Yeast Mutants with Increased Mitochondrial Respiratory Chain and Nitric Oxide Levels

“…We next determined whether the activities of major NO detoxification enzymes were increased by CR. Yeast flavohemoglobin (Yhb1), which catalyzes the conversion of NO to nitrite or nitrous oxide, and GSNO reductase (GSNOR, Sfa1) are the two major enzymes that protect yeast cells from nitrosative stress [40, 41, 46]. Interestingly, Yhb1 activity determined in the crude cell lysate [41] was increased by CR (Figure 1(d)), whereas Sfa1 activity [40] was not increased by CR, suggesting that Yhb1 may play an important role in CR.…”

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme

“…Heme b (aka Protoheme IX) is utilized as a cofactor by several mitochondrial proteins/complexes ( Figure 3). These include mitochondrial matrix-localized flavohemoglobin Yhb1 (neuroglobin in mammals) [6,109], IMM-anchored ETC enzymes ubiquinol-cytochrome c oxidoreductase (complex III, bc 1 complex) [110] and succinate dehydrogenase (complex II) [6,111,112]. Additionally, yeast mitochondria harbor several fungi-specific hemoproteins such as CcO assembly factor Mss51 [113], cytochrome c peroxidase Ccp1 [114] and L-lactate cytochrome c oxidoreductase Cyb2 [115].…”