Mutant strains of the filamentous cyanobacterium Anabaena strain PCC 7120 ΔHup (dc-Q193S and dc-R284H) with amino acid substitutions located in the vicinity of the FeMo cofactor of nitrogenase possess nitrogenase activity with a hydrogen production rate of approximately 18 μmol Н2/(mg h), which is ~30% lower than that of the parental strain ΔHup. The photosynthetic activity of mutants is also reduced. Unlike the parental strain ΔHup, the dc-Q193S mutant shows a lower temperature optimum for hydrogen photoproduction. This difference is probably due to the lowered filament strength (fragmentation). Hydrogen photoproduction in mutants does not significantly differ from that of the parental strain in relation to the activation energy, light saturation constants (41–62 μmol quanta /(m2 s)), and acetylene-induced inhibition. However, in contrast to the parental strain, hydrogen photoproduction in the mutant strains is not inhibited by molecular nitrogen, i.e., amino acid substitutions cause significant changes in the reaction requiring eight electrons (N2 fixation). The possibility to use nitrogen or atmospheric air instead of argon in the hydrogen production is promising from the practical point of view, though reduced activity and increased fragility of filaments in the studied mutants limit the possibility of their practical use.